CRZ1 MUTANT FUNGAL CELLS

This Application claims the benefit of U.S. Provisional Patent Application No.

61/716,670, filed October 22, 2012; which is herein incorporated by reference in its entirety. Field of the Invention

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.

Background of the Invention

GlycoFi has engineered Pichia to produce recombinant glycoproteins with humanlike glycosylation. However, 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. For example, Candida albicans ochl knock-outs are temperature sensitive at 42°C (Bates et a/., 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 ochl knock-outs are temperature sensitive at 45°C (Kim et a/., Functional Characterization of the Hansenula polymorpha HOC1 , OCH1 , and OCR1 Genes as Members of the Yeast OCH1 Mannosyltransferase Family Involved in Protein

Glycosylation, The Journal of Biological Chemistry, 281 : 6261 -6272 (2006));

Schizosaccharomyces pombe ochl knock-outs are temperature sensitive at 37°C (Yoko-o et a/., Schizosaccharomyces pombe och1 ⁺ encodes alpha-1 ,6-mannosyltransferase that is involved in outer chain elongation of N-linked oligosaccharides, FEBS Letters 489: 75-80 (2001 )); Saccharomyces cerevisiae ochl knock-outs are temperature sensitive at 37°C (Nakayama et al., OCH1 encodes a novel membrane bound mannosyltransferase: outer chain elongation of asparagine-linked oligosaccharides, EMBO J. 11 (7):251 1-9 (1992)); and Pichia pastoris ochl knock-outs are temperature sensitive at 37°C (Choi et a/., Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris, Proc Natl Acad Sci U S A. 100(9):5022-7 (2003)). Additional genetic modifications to make ochT fungal host cells {e.g., Pichia cells) more robust in cell culture would be of value.

An unlikely candidate for genetic modification in order to increase Pichia culture robustness is CRZ1, a zinc finger transcription factor. CRZ1 is known to regulate a number of S. cerevisiae plasma membrane and cell wall regulatory genes (Cyert, Biochemical and Biophysical Research Communications 31 1 :1 143-1 150 (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 a/., Genes & Development 1 1 :3445-3458 (1997); Stathopoulos et a/., Genes & Development 1 1 : 3432-3444 (1997)).

Summary of the Invention

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. In an embodiment of the invention, (i) 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; C41 1 ^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 CRZ1 does not encode a functional C-terminal zinc-finger domain. In an embodiment of the invention, the isolated fungal host cell also comprises one or more (e.g., any 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 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); (v) wherein an endogenous dolichol-P-Man dependent alpha-1 ,3-mannosyltransferase (e.g., ALG3) is mutated, disrupted, truncated or partially or fully deleted; (vi) comprising a polynucleotide encoding an endomannosidase; (vii) comprising one or more polynucleotides encoding a bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase, an N- acetylneuraminate-9-phosphate synthase, or a CMP-sialic acid synthase; (viii) wherein an endogenous ATT1 gene is mutated, disrupted, truncated or partially or fully deleted; (ix) wherein an alpha-1 ,6-mannosyltransferase (e.g., OCH1) is mutated, disrupted, truncated or partially or fully deleted; (x) comprises a galactosyltransferase e.g., an alpha 1 , 3- galactosyltransferase or a beta 1 ,4- galactosyltransferase; (xi) comprises a nucleotide sugar transporter, e.g., UDP-Galactose transporter (DmUGT); (xii) comprises a sialyltransferase, e.g., alpha-2,6-sialyl transferase (MmST6-33); (xiii) comprises a acetylglucosaminyl transferase, e.g., GNT1 or GNT2 or GNT4; and/or (xiv) wherein one or more endogenous protease genes (e.g., PEP4 and PRB1) are mutated, disrupted, truncated or partially or fully deleted. 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; C41 1 ^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 _C rz7 ^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; C41 1 ^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 _C rz1 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. In an embodiment of the invention, 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. Brief Description of the Figures

Figure 1 : Temperature resistant mutants displaying improved fermentation robustness and Fc titer.

Figure 2(a-c): Lineages of mutagenized Pichia pastoris strains.

Figure 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; A1 H= 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₊ .

Figure 4: Deep sequencing of temperature-resistant mutants identified multiple mutations in Pp02g02120 (CRZ1) and two mutations in Pp01g00680 (ATT1), corresponds to circled "+" symbols.

Figure 5: Mutated protein Pp02g02120 was homologous to Saccharomyces cerevisiae CRZ1 zinc finger transcription factor involved in stress responses. The various isolated mutant PpCRZI alleles are shown. The "*" shows the location of the indicated mutation.

Figure 6: Plasmid pGLY12829.

Figure 7: Plasmid pGLY12832.

Figure 8: DNA Constructs constructed for Pichia pastoris CRZ1 deletion and truncations.

Figure 9: Pichia pastoris CRZ1 deletion and truncation mutants exhibited improved fermentation robustness at 32°C.

Figure 10: Pichia pastoris CRZ1 deletion further improves fermentation robustness in an attl mutant strain at 34°C. Detailed Description of the Invention

Contrary to what was expected, Pichia pastoris cells, lacking functional CRZ1 , exhibited enhanced temperature-resistance and increased robustness.

To broadly improve strain quality, random mutagenesis was conducted and several temperature-resistant mutant Pichia pastoris strains with significantly improved fermentation robustness were identified. Whereas the non-mutagenized glycoengineered parental strains display a temperature-sensitive phenotype (Choi et al. 2003) and are viable for 40 to 60 hours after induction at 32°C, the mutants all lasted between 90 to 1 10 hours after induction at 32°C. This extended induction period significantly increased the yield and quality of recombinant proteins expressed from these temperature-resistant strains. To uncover the mutations responsible for this increased thermal tolerance and fermentation robustness, genome-sequencing for 9 independently isolated mutants was performed, and non-synonymous mutations within distinct open reading frames (ORF) per mutant were identified. Remarkably, all 9 mutants contained distinct mutations within the coding region of one gene, Pichia pastoris CRZ1. More importantly, the Pichia pastoris CRZ1 mutation was the only non-synonymous single-nucleotide-variation (SNV) detected in three mutants YGLY29010, YGL29031 , and YGL29042. Collectively, these genome-sequencing results show that the mutations within the Pichia pastoris CRZ1 gene were responsible for the temperature-resistance and fermentation robustness phenotypes. Moreover, non- mutagenized glyco-engineered strains in which endogenous CRZ1 was mutated, and, thus, lacked functional CRZ1 protein, similarly exhibited viability for 90-1 10 hours after induction at 32°C.

A "CRZ1 ^wt fungal host cell comprises a wild-type CRZ1.

"PpCRZI " is Pichia pastoris CRZ1.

"ScCRZI " is Saccharomyces cerevisiae CRZ1 .

High temperature with respect to the growth of isolated fungal cells such as Pichia, e.g., Pichia pastoris, 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. For example, 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.

An "endogenous" gene is a chromosomal copy of the gene.

A glossary of gene names that may be mentioned herein is as follows:

ScSUC2 S. cerevisiae Invertase

OCH1 Alpha- 1 ,6-mannosyltransferase

K1MNN2-2 . lactis UDP-GlcNAc transporter nucleotide sugar transporter BMT1 Beta-mannose-transfer (beta-mannose elimination) beta-mannosyltransferase

BMT2 Beta-mannose-transfer (beta-mannose elimination)

BMT3 Beta-mannose-transfer (beta-mannose elimination)

BMT4 Beta-mannose-transfer (beta-mannose elimination)

MNN4L1 MNN4-like 1 (charge elimination)

MmSLC35A3 Mouse homologue of UDP-GlcNAc transporter

Phosphomannosylation of N-linked oligosaccharides

PNOl

(charge elimination)

MNN4 Mannosyltransferase (charge elimination)

ScGALlO UDP -glucose 4-epimerase

XB33 Truncated HsGalTl fused to ScKRE2 leader

DmUGT UDP-Galactose transporter

KD53 Truncated DmMNSII fused to ScMNN2 leader

TC54 Truncated RnGNTII fused to ScMNN2 leader

NA10 Truncated HsGNTI fused to PpSEC12 leader

FB8: Truncated MmMNSl A fused to ScSEC12 leader

MmCST Mouse CMP-sialic acid transporter

Human UDP-GlcNAc 2-epimerase/N-

HsGNE

acetylmannosamine kinase

HsCSS Human CMP-sialic acid synthase

HsSPS Human N-acetylneuraminate-9-phosphate synthase

Truncated Mouse alpha-2,6-sialyl transferase fused

MmST6-33

to ScKRE2 leader

TrMDSl Secreted T. reseei MNS1

STE13 Golgi dipeptidyl aminopeptidase

DAP2 Vacuolar dipeptidyl aminopeptidase

dolichol-P-Man dependent alpha(l-3)

ALG3

mannosyltransferase

STT3D oligosaccharyltransferase

CiMNSl Coccidioides immitis mannosidase I Molecular Biology

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., James M. Cregg (Editor), Pichia Protocols (Methods in Molecular Biology), Humana Press (2010), Sambrook et al. Molecular Cloning: A

Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing

Associates (1992, and Supplements to 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ. Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, N.J.; Handbook of Biochemistry: Section A Proteins, Vol I, CRC Press (1976); Handbook of Biochemistry: Section A Proteins, Vol II, CRC Press (1976); Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999), Animal Cell Culture (R.I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984).

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., _C rz7 ^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). As used herein, the term "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 "protein", "peptide" or "polypeptide" (e.g., a heterologous polypeptide such as an immunoglobulin heavy chain and/or light chain) includes a contiguous string of two or more amino acids. Any polypeptide comprising an amino acid sequence set forth herein (e.g., Crz7 ^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.

The term "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., crz7 ^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.

"Amplification" of DNA as used includes the use of polymerase chain reaction (PCR) to increase the concentration of a particular DNA sequence within a mixture of DNA sequences. For a description of PCR see Saiki, et a/., Science (1988) 239:487.

In general, 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. Crz7 ^mutant polynucleotide operably linked to a promoter forms part of the present invention. Also, an isolated crz 7 ^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.

The present invention includes vectors or cassettes which comprise _C rz7 ^mutant polynucleotide. Vectors containing a heterologous polynucleotide encoding a heterologous polypeptide can also be used in various _C rz7 ^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 a/., 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, MA. Such vectors optionally include a secretion signal (e.g., alpha-mating factor (a-MF) pre-pro leader sequence) operably linked to a heterologous polynucleotide. Also, an isolated _C rz7 ^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. The term "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.

The term 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 _crz i ^mutani 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 _C rz7 ^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. The following references regarding the BLAST algorithm are herein incorporated by reference: 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. (1993) 17:149-163; Hancock, J.M., et al., Comput. Appl. Biosci. (1994) 10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M.O., et al, "A model of evolutionary change in proteins." in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. M.O. Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, DC; Schwartz, R.M., et al., "Matrices for detecting distant relationships." in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3." M.O. Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, DC; Altschul, S.F., J. Mol. Biol. (1991 ) 219:555-565; States, D.J., et al., Methods (1991 ) 3:66-70; Henikoff, S., et al., Proc. Natl. Acad. Sci. USA (1992)89:10915-10919; Altschul, S.F., et al., J. Mol. Evol. (1993) 36:290-300; ALIGNMENT STATISTICS: Karlin, S., et al., Proc. Natl. Acad. Sci. USA (1990) 87:2264-2268; Karlin, S., et al, Proc. Natl. Acad. Sci. USA (1993) 90:5873-5877; Dembo, A., et al, Ann. Prob. (1994) 22:2022-2039; and Altschul, S.F.

"Evaluating the statistical significance of multiple distinct local alignments." in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp. 1-14, Plenum, New York.

CRZ1

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). Specific examples of 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. These 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; C41 1 ->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 HEDLHSGERKYCCKGVLSDGVTTWGCEKRFARTDALGRHF TECGKLC (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:

ScCRZl (1

PpCR l (1

ScCRZl (51

PpCRZl (21

101 150

ScCRZl (101 A¾LNYQS;L¾KPD|PQGIQLQQQQLQ|QQQQQQQ¾QI3QQKQTPTLKVEQSD

PpCRZl (58 TSSFST¾S¾QEI|P SVKIKLEFD§D¾PVFYQES¾--P

151 .. 00

ScCRZl (151 FQllSDIljSPAblQHR^SLlNgFLSPRS YDlTl^-SGIDS YSDrESN

PpCRZl (95 ~~VP¾KH!J V D||ETR: |AQDFNQYLNAtgv|R|NSISNLSELST¾S¾

201 250

ScCRZl (200 YHT|YljYPQDLVSSPAiSH|TiNNDDFDDLLHASMH YLLPV|SHGYK

PpCRZl (140 ITPB ^! Tl:iLHDQASLSPi¾LsiN|DER EL ¾EHQLDHSQPYV ilK EA

251 300

ScCRZl (250 HISN ^' i ELDDLtSLTYSD NiLSASN|siF S¾S!GIINTADTQNSTIAI

PpCRZl (190 AYEEESEiJHHRLERLTET¾L|HQDQL§L¾QjEQ§ QT

301 B 350

ScCRZl (300 K|KVGTNQK§LI,TIΡ/ΓSST^^

PpCRZl (227 PH| LSPpiQliQTP;iIKVLQAPNDI^N¾ESlFSQSNHSSP|NTPKHS

351 400

ScCRZl (350 DGTLQLKVRDNESYSATNNNNLL ^: RPDPNDfNNEA|S;D|DR|FEDHN|RK

PpCRZl (274 R s -MsSsSNDRcjfDIPQ|S'S|LD jsSF|§PSDQ

401 B B 450

ScCRZl (400 LKLKfSRRRSSQfSNNSFTS|RSS|3RSISPDE;KA¾SISANREKLLEMAD

PpCRZl (306 FQA |EGi¾QRR gEStJgRN¾|ER¾lSR ^' PP£SR§RSR

451 500

ScCRZl (450 LLPSSENDNNRERYDN])SKTS NTINSfiNFNEDNNNNN¾LTSKg IESGI

PpCRZl (343 DSATD|HMEVMS' REKTLEliAASQE

501 550

ScCRZl (500 VNIKNELDDTSKDLGILLDIDSLGQFEQ VGFKNDDNHENNDNGTFSV K

PpCRZl (367 S_

ScCRZl (550

PpCRZl (368

ScCRZl (600

PpCRZl (409

ScCRZl (649) T.GRIIFRTF.SGj|RCJTI ^' 'T,Y§F,A¾QB".KSGQES

PpCRZl ( 459 ) LGRHFK ECG^LCIKPLM¾EL§REE§YRRNEPVTEMNDELYSQSVQDIFS

701

ScCRZl (679)

PpCRZl (509) SQRLGQNIDD

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 ^Λ in the BLASTP comparison shown above

The identity of CRZ1 is known in the art. Specific examples of CRZ1 are set forth below. In an embodiment of the invention, 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. In an embodiment of the invention, 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 polypeptide

msfsngnmas ymtssngeeq sinnkndidd nsayrrnnfr nssnsgshtf qlsdldldvd mrmdsansse kisknlssgi pdsfdsnvns llspssgsys adlnyqslyk pdlpqqqlqq qqqqqqqqqq qqqqqqkqtp tlkveqsdtf qwddiltpad nqhrpsltnq flsprsnydg ttrssgidsn ysdtesnyht pylypqdlvs spamshltan nddfddllsv asmnsnyllp vnshgykhis nldelddlls ltysdnnlls asnnsdfnns nngiintadt qnstiainks kvgtnqkmll tiptsstpsp sthaapvtpi isiqefnegh fpvkneddgt lqlkvrdnes ysatnnnnll rpddndynne alsdidrsfe diingrklkl kksrrrssqt snnsftsrrs srsrsispde kaksisanre kllemadllp ssendnnrer ydndsktsyn tinssnfned nnnnnlltsk pkiesgivni knelddtskd lgilldidsl gqfeqkvgfk nddnhenndn gtfsvkkndn lekldsvtnn rknpanfacd vcgkkftrpy nlkshlrtht nerpficsic gkafarqhdr krhedlhtgk kryvcggklk dgkpwgcgkk farsdalgrh fktesgrrci tplyeearqe ksgqes

(SEQ ID NO: 1 )

Pichia pastoris CRZ1 wild-type open reading frame

ATGGCAGACCAACGGCTTGAGGATGAGTTTGATATCTCCAGATACTTATCTATTTCTCCT ATCGAGT CAGCTTCAATCGAAGAATCAATCAACGGTTTAATGAGTAGTTGGATTCCTCCGGCTAAGG GTGAGAT TAGAGATTCACTTCCTCCAAACGCTTCTTTTGAAGCTACAGACAGTTTTTCAACCAGTTC ATACCAG GAAATTATACCAGCACAGGTGAAAATAAAACTGGAGTTTGATAATGACCAGCAGCCTGTT TTCTATC AAGAATCGCAACCAGTTTATGATAAGCATTTAACCGTCAATGATCAGGAAACCAGAAGCG CCCAAGA CTTCAACCAATACTTGAATGCTGATGCCGTATCGAGGACCAACTCCATCTCCAACTTATC GGAGCTG TCAACTCATTCCCATATTACCCCTCCAACGCTACTTCATGATCAAGCCTCATTGTCTCCT GCTCTCT TATCTATGAACAGTGATGAAAGAAACGAACTCAATCTGGAAACACTACAGCTAGATCAAA CGTCACA GCCTTACGTGAATCAGATAAAAACGGAGGCAGCTTACGAAGAGCTTTCAGAGTTACACCA CAGATTA GAAAGACTCACTGAGACAAATTTAATTCATCAAGACCAGCTTCAACTCGAACAACAAGAG CAACAAA ATCAGACTCCTCATACTCTCAGTCCTCCTATACAACTTCAGACTCCCATAATCAAAGTCT TGCAAGC CCCAAATGATATAGCAGCAAATACCCCGTCTCTTTTTTCTCAATCTAACCATTCATCTCC ATATAAC ACACCCAAACATTCCAGGTCAAACTCGTTGAGTTCAAA GACAGACAACATGATATTCCACAAATAT CCTCAGTTTTAGACACGTCTTCGTTTTTGGTACCTGGAGATCAGTTTCAAGCAATGAGAG AAGGTAG ACAGAGGAGGAAATCCGAGTCCAACTCTAGAAACTCGAAAGAACGTTCTAAATCTAGGGA ACCGCCA AAGTCCAGGTCTAGATCTAGAGACAGTGCAACAGATCATCATATGGAAGTTATGAGCAGA GAAAAGA CTCTTGAGTTGGCAGCTTCTCAGCCAAGCTCCAAGACGCCGCAAAAGAACCCTTCCATCT ATGCTTG CTCGCTCTGCTCCAAGAGATTCACAAGACCATATAATTTGAAGTCTCACCTTCGCACGCA CGCTGAT GAAAGGCCTTTCCAGTGTTCAATATGCGGGAAGGCATTTGCTCGTTCTCACGACAGAAAG CGTCATG AGGATCTGCATAGTGGTGAACGAAAGTATTGCTGCAAAGGTGTTTTGTCTGACGGAGTAA CTACATG GGGCTGTGAAAAAAGATTTGCCCGAACAGATGCGCTGGGTAGACATTTCAAAACTGAATG TGGTAAA CTGTGTATCAAGCCGCTGATGGATGAACTAAAGAGGGAGGAAGCTTACAGGAGGAATGAA CCAGTAA CAGAAATGAATGACGAGCTTTACTCCCAATCTGTCCAAGATATATTTAGCTCTCAGCGGC TTGGTCA GAACATAGATGACtga

(SEQ ID NO: 2)

Pichia pastoris CRZ1 wild-type polypeptide

MADQRLEDEFDISRYLS ISPIESAS IEES INGLMSSWIPPAKGEIRDSLPPNASFEATDSFSTSSYQEI I PAQVKIKLEFDNDQQPVFYQESQPVYDKHLTVNDQETRSAQDFNQYLNADAVSRTNSISN LSELSTHSHI TPPTLLHDQASLSPALLSMNSDERNELNLETLQLDQTSQPYVNQIKTEAAYEELSELHHR LERLTETNLI HQDQLQLEQQEQQNQTPHTLSPPIQLQTPI IKVLQAPNDIAANTPSLFSQSNHSSPYNTPKHSRSNSLSS NDRQHDIPQISSVLDTSSFLVPGDQFQAMREGRQRRKSESNSRNSKERSKSREPPKSRSR SRDSATDHHM EVMSREKTLELAASQPSSKTPQKNPSIYACSLCSKRFTRPYNLKSHLRTHADERPFQCSI CGKAFARSHD RKRHEDLHSGERKYCCKGVLSDGVTTWGCEKRFARTDALGRHFKTECGKLCIKPLMDELK REEAYRRNEP VTEMNDELYSQSVQDIFSSQRLGQNIDD*

(SEQ ID NO: 3)

Host Cells

The present invention includes isolated _C rz7 ^mutant fungal host cells which may include additional mutations in its genetic background. In a " _crz 7 ^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. In an embodiment of the invention, the _C rz7 ^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 90-1 10 hours of induction at 32°C. In an embodiment of the invention, an isolated _C rz7 ^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 _crz 7 ^mutant fungal host cells are within the scope of the present invention). In an embodiment of the invention, an isolated _C rz7 ^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 _cr z7 ^mutant fungal host cells are within the scope of the present invention).

In an embodiment of the invention, the isolated crz 7 ^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 crz 7 ^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. In an embodiment of the invention, in an isolated _C rz7 ^mutant fungal host cell of the present invention, endogenous CRZ1 is mutated such that it lacks a functional C-terminal zinc-finger domain, e.g., due to mutation or truncation. In an embodiment of the invention, 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 _C rz1 ^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; or otherwise inactivated partially or fully in any way whatsoever. Alternatively, the regulatory region of such an endogenous CRZ1 gene may be partially or fully deleted, disrupted or mutated such that no significant amount of functional CRZ1 polypeptide is expressed in the cell. Moreover, 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. Such isolated _C rz1 ^mutant fungal host cells are part of the present invention.

The scope of the present invention encompasses isolated _C rz7 ^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. _C rz7 ^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. _C rz7 ^mutant fungal host cells exhibited less than a 5 score for lysis for between 90 and 1 10 hours following induction at 32°C. The culture medium containing crz7 ^mutant fungal host cells induced at 32°C had 30 micrograms/milliliter or less double standed DNA for between 90 and 1 10 hours. When the cells lyse, double stranded DNA is released into the medium; thus, double stranded DNA content of the culture is a marker for cell lysis and a means by which to determine cell viability in the culture. 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 culture medium. 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 1 10 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. For example, the present invention includes methods for making one or more heterologous polypeptides in an isolated crz7 ^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 _C rz7 ^mutant fungal host cell and (ii) culturing the _C rz7 ^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 crz7 ^mutant fungal host cell.

In an embodiment of the invention, a _C rz7 ^mutant fungal host cell also comprises a mutation in ATT1. In one embodiment of the invention, a _C rz7 ^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. In an embodiment of the invention, 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 oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum and Neurospora crassa. In one particular embodiment of the invention, an isolated fungal host cell is as discussed above except that the term excludes Saccharomyces cerevisiae.

In an embodiment of the invention, the isolated fungal host cell is glycoengineered. In an embodiment of the invention, 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, PN01, 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. For example, in an embodiment of the invention, a glycoengineered isolated fungal host cell comprises any one or more 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 enzyme;

(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);

(v) wherein an endogenous dolichol-P-Man dependent alpha-1 ,3-mannosyltransferase (e.g., Alg3) is mutated, disrupted, truncated or partially or fully deleted;

(vi) comprising a polynucleotide encoding an endomannosidase;

(vii) comprising one or more polynucleotides encoding a bifunctional UDP-N- acetylglucosamine-2-epimerase/N-acetylmannosamine kinase, an N-acetylneuraminate-9- phosphate synthase, or a CMP-sialic acid synthase;

(viii) wherein endogenous ATT1 gene is mutated, disrupted, truncated or partially or fully deleted;

(ix) wherein an endogenous alpha-1 ,6-mannosyltransferase (e.g. 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. 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.

As used herein, the terms "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)).

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). 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). 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).

In an embodiment of the invention, an isolated crz7 ^mutant fungal host cell, such as a Pichia cell (e.g., Pichia pastoris), is genetically engineered to include a nucleic acid that encodes an a-1 ,2-mannosidase that has a signal peptide that directs it for secretion. For example, in an embodiment of the invention, the _C rz1 ^mutant host cell is engineered to express an exogenous a-1 ,2-mannosidase enzyme having an optimal pH between 5.1 and 8.0, preferably between 5.9 and 7.5. In an embodiment of the invention, 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. Patent 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 , a-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 _C rz1 ^mutant fungal host cell that does not display alpha-1 ,6 mannosyltransferase (e.g.

OCH1 ) activity with respect to the N-glycan on a glycoprotein, the method comprising the step of introducing into the _C rz1 ^mutant ochl ^" 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 the ER or Golgi apparatus of the host cell, in excess of 30 mole % of the N-glycan structures attached thereto have a Man ₅ GlcNAc2 glycoform that can serve as a substrate for GlcNAc transferase I in vivo.

Isolated _C rz1 ^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 β-mannosyltransferase genes (e.g., BMTI, BMT2, BMT3, and/or BMT4) (See, U.S. Patent 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. 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 , β-mannosyltransferase ^" (e.g., bmtT, bmtZ, bunts ^' , 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.

Isolated _C rz1 ^mutant fungal host cells (e.g., Pichia, e.g., Pichia pastoris) of the present invention 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 PN01 and MNN4B (See for example, U.S. Patent 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. In an embodiment of the invention, 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 ₍ i- 4)Man ₃ GlcNAc ₂ , NANA^^GIcNAc^MansGlcNAc;?, and NANA ₍ i-4)Gal ₍ i-4)Man ₃ GlcNAc2; 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 invention, selected from the group consisting of Man ₆ GlcNAc ₂ , Man ₇ GlcNAc ₂ ,

Mang ₈ 1 cNAc ₂ , and Man ₉ GlcNAc ₂ . 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 ^mulanl ,

phosphomannosyl transferase ^" (e.g., pno 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.

Isolated _C rz1 ^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 WO201 1/06389. 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 _C rz1 ^mutant fungal host cells (e.g., Pichia pastoris) of the present invention 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 _C rz1 ^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. 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 _C rz1 ^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 ₍ i _-4) GlcNAc ₍ i _-4) Man ₃ GlcNAc ₂ , e.g., by a method comprising: (a) transforming, into the _C rz1 ^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 polynucleotide encoding a CMP-sialic acid transporter; and (c) transforming into the host cell a polynucleotide molecule encoding a 2,6-sialyltransferase catalytic domain fused to a cellular targeting signal peptide, e.g., encoded by nucleotides 1- 108 of the S. cerevisiae Mnn2; wherein, upon passage of a recombinant glycoprotein through the secretory pathway of the host cell, a recombinant sialylated glycoprotein comprising a glycoform selected from the group consisting of NANA _(1-4) Gal(i -4 ₎ GlcNAC(i - 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.

In addition, isolated czr7 ^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. In one embodiment of the present invention, the isolated _C zr1 ^mutant fungal host cell comprises an isolated nucleic acid molecule encoding β-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 GlcNAcpi ,2-Mana1 ;

GlcNAcpi ,4-Mana1 ,3, GlcNAcpi ,2-Mana1 ,6, GlcNAcpi ,4-Mana1 ,6 and GlcNAcpi ,6- Mana1 ,6; wherein the host cell is diminished or depleted in dolichyl-P-Man:Man ₅ GlcNAc2- PP-dolichyl a-1 ,3 mannosyltransferase activity, and wherein the host cell produces a glycoprotein having one or more galactose residues. 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.

In an embodiment of the invention, an isolated _C zr1 ^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 , ochT 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 _C rz1 ^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. 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 _C rz1 ^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 _C rz1 ^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 ^{muta t} , 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 _C rz1 ^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. 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. As used herein, the term "essentially free of 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. Expressed in terms of purity, 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.

As used herein, a 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. For example, in an embodiment of the present invention, 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. Thus, the term "essentially free of fucose" encompasses the term

"lacking fucose." However, 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.

Protein Expression

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 crz7 ^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. In an embodiment of the invention, fungal host cells can be grown in liquid culture medium in shaken-flasks or in fermentors (e.g., 1 L, 2L, 5L, 10L, 20L, 30L, 50L, 100L, 200L, 500L, 1000L, 10,000L volume). Various growth mediums may be used to culture fungal host cells. In an embodiment of the invention, 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. In an embodiment of the invention, the temperature is maintained at about 24°C or 26°C or 28°C or 30°C or 32°C or 34°C. In an embodiment of the invention, dissolved oxygen in the growth medium is maintained at about 20% or 30%. In an embodiment of the invention, the growth medium contains yeast nitrogen base (e.g., with ammonium sulfate; with or without essential amino acids), 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). In an embodiment of the invention, the growth medium is minimal medium containing yeast nitrogen base, water, a carbon source such as dextrose, methanol or glycerol, biotin and histidine. In an embodiment of the invention, the cell culture comprises trace

minerals/nutrients such as copper, iodine, manganese, molybdenum, boron, cobalt, zinc, iron, biotin and/or sulfur, e.g., CuS0 ₄ , Nal, MnS0 ₄ , Na ₂ Mo0 ₄ , H ₃ B0 ₃ , CoCI ₂ , ZnCI ₂ , FeS0 ₄ , biotin and/or H ₂ S0 ₄ . In an embodiment of the invention, 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 CAz7 ^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,

(i) in a batch phase (e.g., a glycerol batch phase) wherein the cells are grown with a non- fermentable carbon source, such as glycerol, e.g., until the non-fermentable carbon source is exhausted;

(ii) in a batch-fed phase (e.g., a glycerol batch-fed phase) wherein additional non- fermentable carbon source (e.g., glycerol) is fed, e.g., at a growth limiting rate; and

(iii) in a methanol fed-batch phase wherein the cells are grown in the presence of methanol and, optionally, additional glycerol.

In an embodiment of the invention, in the methanol fed-batch phase, 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).

In an embodiment of the invention, prior to the batch phase, an initial seed culture is grown to a high density (e.g., OD ₆₀ o of about 2 or higher) and the cells grown in the seed culture are used to inoculate the initial batch phase culture medium.

In an embodiment of the invention, after the batch-fed phase and before the methanol fed-batch phase, 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. For example, 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. Examples

This section is intended to further describe the present invention and should not be construed to further limit the invention. Any composition or method set forth herein constitutes part of the present invention.

Example 1 : Identification of CRZ1 mutations.

Experimental Methods

UV mutagenesis, fed-batch fermentations, IgG purifications, N-glycan

characterizations, as well as all other analytical assays, were performed as previously described (Barnard et al. 2010; Jiang et al. 201 1 ; 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.

Result and Discussion

Temperature-Resistant Mutants Displayed Substantially Enhanced

Fermentation Robustness. To identify Pichia host strains with increased fermentation robustness, we UV-mutagenized temperature-sensitive glyco-engineered strains

(YGLY12905, YGLY22835, and YGLY27890), and selected for temperature-resistant mutants, with the rationale that certain 2 ^nd -site mutations suppressing the temperature- sensitive defect might also compensate for the cell robustness deficiency. After confirming their temperature-resistant phenotypes, these mutants were fermented using standard MeOH fed-batch runs in 1 L DasGip Bioreactors. After an extensive fermentation screening campaign, we identified 9 mutants displaying much enhanced cell robustness during the fermentation process. As shown in Figure 1 , the fermentation process for the non- mutagenized control strain had to be terminated, due to excessive cell lysis, at

approximately 48 hours of induction at 32°C. In contrast, the mutants all displayed significantly improved fermentation robustness. YGLY29010, YGLY29030, YGLY29031 , and YGLY29012 were able to ferment approximately 90 hours; YGLY28997, YGLY28998, YGLY17159, YGLY28999, and YGLY29042 all lasted for more than 100 hours induction at 32°C. Protein Productivity and N-glycan Quality Assessments of the Temperature- Resistant Mutants. Five of the temperature-resistant mutants (YGLY29010, YGLY29030, YGLY29031 , YGLY29042, and YGLY29012) were derived from YGLY27890 (Figure 2), which expresses a human Fc fragment. To evaluate what impacts these temperature- resistant mutations had on Fc productivity and N-glycan quality, we purified the Fc fragments from the 32°C 1 L bioreactors, quantified the broth titer (Figure 1 ), and analyzed the N-glycan profiles (Figure 3) of four temperature-resistant mutants, as well as their un- mutagenized parent strain YGLY27890. Compared with the parental control, four mutants (YGLY29010, YGLY29030, YGLY29031 , and YGLY29042) displayed substantial increases in product titers: in fact, YGLY29042 and YGLY29010 actually secreted approximately 4-5 fold more Fc product. In contrast, the product titer from YGLY29012 was only

approximately 50% of the control strain's titer. For N-glycans derived from the Fc product, we did not observe any significant alterations in the N-glycan profiles (Figure 3). Just like the control strain YGLY27890 (83% A2 and 4% A1 ), all five mutants were able to effectively modify their Fc N-glycans with high levels of terminal sialic acids, with A2 levels ranging from 77 to 84%, and A1 levels from 4 to 7%. Collectively, these results demonstrated that the UV-induced mutations acquired by YGLY29010, YGLY29030, YGLY29031 , and YGLY29042 did not negatively affect their capabilities for producing heterologously expressed human Fc fragment, nor did the mutations resulted in noticeable deteriorations in N-glycan quality.

Genome Sequencing to Identify the Causative Mutation(s) Responsible for the Enhanced Thermal-tolerance and Fermentation Robustness. In order to better understand the molecular mechanisms involved in maintaining cell robustness during fermentation, we sequenced the genomes of these 9 temperature-resistant mutants, as well as two un-mutagenized empty host strains YGLY22812 and YGLY22835. After genome- wide comparisons between the mutants and the un-mutagenized strains, we identified between 1 to 7 non-synonymous mutations (indicated by a "+" Figure 4) in each of these 9 mutants. Three mutants, YGLY29010, YGLY29031 , and YGLY29042, contained a single mutation within a gene, Pp02g02120, which showed a high-level of sequence homology to the CRZ1 gene of Saccharomyces cerevisiae. Remarkably, distinct mutations in the same PpCRZI gene were also detected in YGLY28997, YGLY28998, YGLY17159, YGLY28999, YGLY29030, and YGLY29012. ScCRZI is a calcineurin-responsive zinc finger transcription factor involved in stress responses. The zinc finger domains are very well conserved between PpCRZI and ScCRZI , and are both located at the c-terminal end (see sequence alignment above). As illustrated in Figure 5, 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 PpCRZI fragments without the zinc-finger domain. For 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. The findings that 9 independently isolated temperature-resistant mutants contained different non-sense or mis-sense mutation within the PpCRZI gene strongly suggested that these CRZ1 mutations were causative for the temperature-resistant and increased fermentation robustness phenotypes. Furthermore, 2 of the mutants (yGLY17159 and yGLY28998) also harbored additional mutations in PpATTI , which is a gene previously shown to play critical roles in temperature-tolerance and cell robustness as well. The fact that both yGLY17159 and yGLY28998 were among the most robust mutants isolated suggested the combination of ATT1 and CRZ1 mutations might have additive or synergistic effects for increasing thermal-tolerance and fermentation robustness in glyco-engineered Pichia strains.

PpCRZI Sequences.

1. Pichia pastoris CRZ1 wild-type open reading frame

ATGGCAGACCAACGGCTTGAGGATGAGTTTGATATCTCCAGATACTTATCTATTTCT CCTATCGAGT CAGCTTCAATCGAAGAATCAATCAACGGTTTAATGAGTAGTTGGATTCCTCCGGCTAAGG GTGAGAT TAGAGATTCACTTCCTCCAAACGCTTCTTTTGAAGCTACAGACAGTTTTTCAACCAGTTC ATACCAG GAAATTATACCAGCACAGGTGAAAATAAAACTGGAGTTTGATAATGACCAGCAGCCTGTT TTCTATC AAGAATCGCAACCAGTTTATGATAAGCATTTAACCGTCAATGATCAGGAAACCAGAAGCG CCCAAGA CTTCAACCAATACTTGAATGCTGATGCCGTATCGAGGACCAACTCCATCTCCAACTTATC GGAGCTG TCAACTCATTCCCATATTACCCCTCCAACGCTACTTCATGATCAAGCCTCATTGTCTCCT GCTCTCT TATCTATGAACAGTGATGAAAGAAACGAACTCAATCTGGAAACACTACAGCTAGATCAAA CGTCACA GCCTTACGTGAATCAGATAAAAACGGAGGCAGCTTACGAAGAGCTTTCAGAGTTACACCA CAGATTA GAAAGACTCACTGAGACAAATTTAATTCATCAAGACCAGCTTCAACTCGAACAACAAGAG CAACAAA ATCAGACTCCTCATACTCTCAGTCCTCCTATACAACTTCAGACTCCCATAATCAAAGTCT TGCAAGC CCCAAATGATATAGCAGCAAATACCCCGTCTCTTTTTTCTCAATCTAACCATTCATCTCC ATATAAC AC AC C C AAAC AT T C C AGG T C AAAC T C GT T GAGTTCAAA G AC AG AC AAC AT GAT AT T C C AC AAAT AT CCTCAGTTTTAGACACGTCTTCGTTTTTGGTACCTGGAGATCAGTTTCAAGCAATGAGAG AAGGTAG ACAGAGGAGGAAATCCGAGTCCAACTCTAGAAACTCGAAAGAACGTTCTAAATCTAGGGA ACCGCCA AAGTCCAGGTCTAGATCTAGAGACAGTGCAACAGATCATCATATGGAAGTTATGAGCAGA GAAAAGA CTCTTGAGTTGGCAGCTTCTCAGCCAAGCTCCAAGACGCCGCAAAAGAACCCTTCCATCT ATGCTTG CTCGCTCTGCTCCAAGAGATTCACAAGACCATATAATTTGAAGTCTCACCTTCGCACGCA CGCTGAT GAAAGGCCTTTCCAGTGTTCAATATGCGGGAAGGCATTTGCTCGTTCTCACGACAGAAAG CGTCATG AGGATCTGCATAGTGGTGAACGAAAGTATTGCTGCAAAGGTGTTTTGTCTGACGGAGTAA CTACATG GGGCTGTGAAAAAAGATTTGCCCGAACAGATGCGCTGGGTAGACATTTCAAAACTGAATG TGGTAAA CTGTGTATCAAGCCGCTGATGGATGAACTAAAGAGGGAGGAAGCTTACAGGAGGAATGAA CCAGTAA CAGAAATGAATGACGAGCTTTACTCCCAATCTGTCCAAGATATATTTAGCTCTCAGCGGC TTGGTCA

GAACATAGATGACtga

(SEQ ID NO: 2) 2. Pichia pastoris mutant CRZ1 isolated from yGLY28997 (encoding the mutation:

L33->STOP, mutated nucleotide is in bold font)

ATGGCAGACCAACGGCTTGAGGATGAGTTTGATATCTCCAGATACTTATCTATTTCTCCT ATCGAGT CAGCTTCAATCGAAGAATCAATCAACGGTTAAATGAGTAGTTGGATTCCTCCGGCTAAGG GTGAGAT TAGAGATTCACTTCCTCCAAACGCTTCTTTTGAAGCTACAGACAGTTTTTCAACCAGTTC ATACCAG GAAATTATACCAGCACAGGTGAAAATAAAACTGGAGTTTGATAATGACCAGCAGCCTGTT TTCTATC AAGAATCGCAACCAGTTTATGATAAGCATTTAACCGTCAATGATCAGGAAACCAGAAGCG CCCAAGA CTTCAACCAATACTTGAATGCTGATGCCGTATCGAGGACCAACTCCATCTCCAACTTATC GGAGCTG TCAACTCATTCCCATATTACCCCTCCAACGCTACTTCATGATCAAGCCTCATTGTCTCCT GCTCTCT TATCTATGAACAGTGATGAAAGAAACGAACTCAATCTGGAAACACTACAGCTAGATCAAA CGTCACA GCCTTACGTGAATCAGATAAAAACGGAGGCAGCTTACGAAGAGCTTTCAGAGTTACACCA CAGATTA GAAAGACTCACTGAGACAAATTTAATTCATCAAGACCAGCTTCAACTCGAACAACAAGAG CAACAAA ATCAGACTCCTCATACTCTCAGTCCTCCTATACAACTTCAGACTCCCATAATCAAAGTCT TGCAAGC CCCAAATGATATAGCAGCAAATACCCCGTCTCTTTTTTCTCAATCTAACCATTCATCTCC ATATAAC ACACCCAAACATTCCAGGTCAAACTCGTTGAGTTCAAA GACAGACAACATGATATTCCACAAATAT CCTCAGTTTTAGACACGTCTTCGTTTTTGGTACCTGGAGATCAGTTTCAAGCAATGAGAG AAGGTAG ACAGAGGAGGAAATCCGAGTCCAACTCTAGAAACTCGAAAGAACGTTCTAAATCTAGGGA ACCGCCA AAGTCCAGGTCTAGATCTAGAGACAGTGCAACAGATCATCATATGGAAGTTATGAGCAGA GAAAAGA CTCTTGAGTTGGCAGCTTCTCAGCCAAGCTCCAAGACGCCGCAAAAGAACCCTTCCATCT ATGCTTG CTCGCTCTGCTCCAAGAGATTCACAAGACCATATAATTTGAAGTCTCACCTTCGCACGCA CGCTGAT GAAAGGCCTTTCCAGTGTTCAATATGCGGGAAGGCATTTGCTCGTTCTCACGACAGAAAG CGTCATG AGGATCTGCATAGTGGTGAACGAAAGTATTGCTGCAAAGGTGTTTTGTCTGACGGAGTAA CTACATG GGGCTGTGAAAAAAGATTTGCCCGAACAGATGCGCTGGGTAGACATTTCAAAACTGAATG TGGTAAA CTGTGTATCAAGCCGCTGATGGATGAACTAAAGAGGGAGGAAGCTTACAGGAGGAATGAA CCAGTAA CAGAAATGAATGACGAGCTTTACTCCCAATCTGTCCAAGATATATTTAGCTCTCAGCGGC TTGGTCA GAACATAGATGACtga

(SEQ ID NO: 2 comprising t98a mutation)

3. Pichia pastoris mutant CRZ1 isolated from vGLY29012 (encoding the mutation:

Q214^STOP, mutated nucleotide is in bold font)

ATGGCAGACCAACGGCTTGAGGATGAGTTTGATATCTCCAGATACTTATCTATTTCT CCTATCGAGT CAGCTTCAATCGAAGAATCAATCAACGGTTTAATGAGTAGTTGGATTCCTCCGGCTAAGG GTGAGAT TAGAGATTCACTTCCTCCAAACGCTTCTTTTGAAGCTACAGACAGTTTTTCAACCAGTTC ATACCAG GAAATTATACCAGCACAGGTGAAAATAAAACTGGAGTTTGATAATGACCAGCAGCCTGTT TTCTATC AAGAATCGCAACCAGTTTATGATAAGCATTTAACCGTCAATGATCAGGAAACCAGAAGCG CCCAAGA CTTCAACCAATACTTGAATGCTGATGCCGTATCGAGGACCAACTCCATCTCCAACTTATC GGAGCTG TCAACTCATTCCCATATTACCCCTCCAACGCTACTTCATGATCAAGCCTCATTGTCTCCT GCTCTCT TATCTATGAACAGTGATGAAAGAAACGAACTCAATCTGGAAACACTACAGCTAGATCAAA CGTCACA GCCTTACGTGAATCAGATAAAAACGGAGGCAGCTTACGAAGAGCTTTCAGAGTTACACCA CAGATTA GAAAGACTCACTGAGACAAATTTAATTCATCAAGACTAGCTTCAACTCGAACAACAAGAG CAACAAA ATCAGACTCCTCATACTCTCAGTCCTCCTATACAACTTCAGACTCCCATAATCAAAGTCT TGCAAGC CCCAAATGATATAGCAGCAAATACCCCGTCTCTTTTTTCTCAATCTAACCATTCATCTCC ATATAAC ACACCCAAACATTCCAGGTCAAACTCGTTGAGTTCAAA GACAGACAACATGATATTCCACAAATAT CCTCAGTTTTAGACACGTCTTCGTTTTTGGTACCTGGAGATCAGTTTCAAGCAATGAGAG AAGGTAG ACAGAGGAGGAAATCCGAGTCCAACTCTAGAAACTCGAAAGAACGTTCTAAATCTAGGGA ACCGCCA AAGTCCAGGTCTAGATCTAGAGACAGTGCAACAGATCATCATATGGAAGTTATGAGCAGA GAAAAGA CTCTTGAGTTGGCAGCTTCTCAGCCAAGCTCCAAGACGCCGCAAAAGAACCCTTCCATCT ATGCTTG CTCGCTCTGCTCCAAGAGATTCACAAGACCATATAATTTGAAGTCTCACCTTCGCACGCA CGCTGAT GAAAGGCCTTTCCAGTGTTCAATATGCGGGAAGGCATTTGCTCGTTCTCACGACAGAAAG CGTCATG AGGATCTGCATAGTGGTGAACGAAAGTATTGCTGCAAAGGTGTTTTGTCTGACGGAGTAA CTACATG GGGCTGTGAAAAAAGATTTGCCCGAACAGATGCGCTGGGTAGACATTTCAAAACTGAATG TGGTAAA CTGTGTATCAAGCCGCTGATGGATGAACTAAAGAGGGAGGAAGCTTACAGGAGGAATGAA CCAGTAA CAGAAATGAATGACGAGCTTTACTCCCAATCTGTCCAAGATATATTTAGCTCTCAGCGGC TTGGTCA GAACATAGATGACtga

(SEQ ID NO: 2 comprising c640t mutation)

4. Pichia pastoris mutant CRZ1 isolated from vGLY29010 (encoding the mutation:

L294^STOP, mutated nucleotide is in bold font)

ATGGCAGACCAACGGCTTGAGGATGAGTTTGATATCTCCAGATACTTATCTATTTCTCCT ATCGAGT CAGCTTCAATCGAAGAATCAATCAACGGTTTAATGAGTAGTTGGATTCCTCCGGCTAAGG GTGAGAT TAGAGATTCACTTCCTCCAAACGCTTCTTTTGAAGCTACAGACAGTTTTTCAACCAGTTC ATACCAG GAAATTATACCAGCACAGGTGAAAATAAAACTGGAGTTTGATAATGACCAGCAGCCTGTT TTCTATC AAGAATCGCAACCAGTTTATGATAAGCATTTAACCGTCAATGATCAGGAAACCAGAAGCG CCCAAGA CTTCAACCAATACTTGAATGCTGATGCCGTATCGAGGACCAACTCCATCTCCAACTTATC GGAGCTG TCAACTCATTCCCATATTACCCCTCCAACGCTACTTCATGATCAAGCCTCATTGTCTCCT GCTCTCT TATCTATGAACAGTGATGAAAGAAACGAACTCAATCTGGAAACACTACAGCTAGATCAAA CGTCACA GCCTTACGTGAATCAGATAAAAACGGAGGCAGCTTACGAAGAGCTTTCAGAGTTACACCA CAGATTA GAAAGACTCACTGAGACAAATTTAATTCATCAAGACCAGCTTCAACTCGAACAACAAGAG CAACAAA ATCAGACTCCTCATACTCTCAGTCCTCCTATACAACTTCAGACTCCCATAATCAAAGTCT TGCAAGC CCCAAATGATATAGCAGCAAATACCCCGTCTCTTTTTTCTCAATCTAACCATTCATCTCC ATATAAC ACACCCAAACATTCCAGGTCAAACTCGTTGAGTTCAAA GACAGACAACATGATATTCCACAAATAT CCTCAGTTTGAGACACGTCTTCGTTTTTGGTACCTGGAGATCAGTTTCAAGCAATGAGAG AAGGTAG ACAGAGGAGGAAATCCGAGTCCAACTCTAGAAACTCGAAAGAACGTTCTAAATCTAGGGA ACCGCCA AAGTCCAGGTCTAGATCTAGAGACAGTGCAACAGATCATCATATGGAAGTTATGAGCAGA GAAAAGA CTCTTGAGTTGGCAGCTTCTCAGCCAAGCTCCAAGACGCCGCAAAAGAACCCTTCCATCT ATGCTTG CTCGCTCTGCTCCAAGAGATTCACAAGACCATATAATTTGAAGTCTCACCTTCGCACGCA CGCTGAT GAAAGGCCTTTCCAGTGTTCAATATGCGGGAAGGCATTTGCTCGTTCTCACGACAGAAAG CGTCATG AGGATCTGCATAGTGGTGAACGAAAGTATTGCTGCAAAGGTGTTTTGTCTGACGGAGTAA CTACATG GGGCTGTGAAAAAAGATTTGCCCGAACAGATGCGCTGGGTAGACATTTCAAAACTGAATG TGGTAAA CTGTGTATCAAGCCGCTGATGGATGAACTAAAGAGGGAGGAAGCTTACAGGAGGAATGAA CCAGTAA CAGAAATGAATGACGAGCTTTACTCCCAATCTGTCCAAGATATATTTAGCTCTCAGCGGC TTGGTCA GAACATAGATGACtga

(SEQ ID NO: 2 comprising .881 g mutation)

5. Pichia pastoris mutant CRZ1 isolated from vGLY29042 (encoding the mutation:

S298^STOP, mutated nucleotide is in bold font)

ATGGCAGACCAACGGCTTGAGGATGAGTTTGATATCTCCAGATACTTATCTATTTCTCCT ATCGAGT CAGCTTCAATCGAAGAATCAATCAACGGTTTAATGAGTAGTTGGATTCCTCCGGCTAAGG GTGAGAT TAGAGATTCACTTCCTCCAAACGCTTCTTTTGAAGCTACAGACAGTTTTTCAACCAGTTC ATACCAG GAAATTATACCAGCACAGGTGAAAATAAAACTGGAGTTTGATAATGACCAGCAGCCTGTT TTCTATC AAGAATCGCAACCAGTTTATGATAAGCATTTAACCGTCAATGATCAGGAAACCAGAAGCG CCCAAGA CTTCAACCAATACTTGAATGCTGATGCCGTATCGAGGACCAACTCCATCTCCAACTTATC GGAGCTG TCAACTCATTCCCATATTACCCCTCCAACGCTACTTCATGATCAAGCCTCATTGTCTCCT GCTCTCT TATCTATGAACAGTGATGAAAGAAACGAACTCAATCTGGAAACACTACAGCTAGATCAAA CGTCACA GCCTTACGTGAATCAGATAAAAACGGAGGCAGCTTACGAAGAGCTTTCAGAGTTACACCA CAGATTA GAAAGACTCACTGAGACAAATTTAATTCATCAAGACCAGCTTCAACTCGAACAACAAGAG CAACAAA ATCAGACTCCTCATACTCTCAGTCCTCCTATACAACTTCAGACTCCCATAATCAAAGTCT TGCAAGC CCCAAATGATATAGCAGCAAATACCCCGTCTCTTTTTTCTCAATCTAACCATTCATCTCC ATATAAC ACACCCAAACATTCCAGGTCAAACTCGTTGAGTTCAAATGACAGACAACATGATATTCCA CAAATAT CCTCAGTTTTAGACACGTCTTAGTTTTTGGTACCTGGAGATCAGTTTCAAGCAATGAGAG AAGGTAG ACAGAGGAGGAAATCCGAGTCCAACTCTAGAAACTCGAAAGAACGTTCTAAATCTAGGGA ACCGCCA AAGTCCAGGTCTAGATCTAGAGACAGTGCAACAGATCATCATATGGAAGTTATGAGCAGA GAAAAGA CTCTTGAGTTGGCAGCTTCTCAGCCAAGCTCCAAGACGCCGCAAAAGAACCCTTCCATCT ATGCTTG CTCGCTCTGCTCCAAGAGATTCACAAGACCATATAATTTGAAGTCTCACCTTCGCACGCA CGCTGAT GAAAGGCCTTTCCAGTGTTCAATATGCGGGAAGGCATTTGCTCGTTCTCACGACAGAAAG CGTCATG AGGATCTGCATAGTGGTGAACGAAAGTATTGCTGCAAAGGTGTTTTGTCTGACGGAGTAA CTACATG GGGCTGTGAAAAAAGATTTGCCCGAACAGATGCGCTGGGTAGACATTTCAAAACTGAATG TGGTAAA CTGTGTATCAAGCCGCTGATGGATGAACTAAAGAGGGAGGAAGCTTACAGGAGGAATGAA CCAGTAA CAGAAATGAATGACGAGCTTTACTCCCAATCTGTCCAAGATATATTTAGCTCTCAGCGGC TTGGTCA GAACATAGATGACtga

(SEQ ID NO: 2 comprising c893a mutation)

6. Pichia pastoris mutant CRZ1 isolated from vGLY28998 (encodinq the mutation:

E403->G, mutated nucleotide is in bold font)

ATGGCAGACCAACGGCTTGAGGATGAGTTTGATATCTCCAGATACTTATCTATTTCT CCTATCGAGT CAGCTTCAATCGAAGAATCAATCAACGGTTTAATGAGTAGTTGGATTCCTCCGGCTAAGG GTGAGAT TAGAGATTCACTTCCTCCAAACGCTTCTTTTGAAGCTACAGACAGTTTTTCAACCAGTTC ATACCAG GAAATTATACCAGCACAGGTGAAAATAAAACTGGAGTTTGATAATGACCAGCAGCCTGTT TTCTATC AAGAATCGCAACCAGTTTATGATAAGCATTTAACCGTCAATGATCAGGAAACCAGAAGCG CCCAAGA CTTCAACCAATACTTGAATGCTGATGCCGTATCGAGGACCAACTCCATCTCCAACTTATC GGAGCTG TCAACTCATTCCCATATTACCCCTCCAACGCTACTTCATGATCAAGCCTCATTGTCTCCT GCTCTCT TATCTATGAACAGTGATGAAAGAAACGAACTCAATCTGGAAACACTACAGCTAGATCAAA CGTCACA GCCTTACGTGAATCAGATAAAAACGGAGGCAGCTTACGAAGAGCTTTCAGAGTTACACCA CAGATTA GAAAGACTCACTGAGACAAATTTAATTCATCAAGACCAGCTTCAACTCGAACAACAAGAG CAACAAA ATCAGACTCCTCATACTCTCAGTCCTCCTATACAACTTCAGACTCCCATAATCAAAGTCT TGCAAGC CCCAAATGATATAGCAGCAAATACCCCGTCTCTTTTTTCTCAATCTAACCATTCATCTCC ATATAAC ACACCCAAACATTCCAGGTCAAACTCGTTGAGTTCAAA GACAGACAACATGATATTCCACAAATAT CCTCAGTTTTAGACACGTCTTCGTTTTTGGTACCTGGAGATCAGTTTCAAGCAATGAGAG AAGGTAG ACAGAGGAGGAAATCCGAGTCCAACTCTAGAAACTCGAAAGAACGTTCTAAATCTAGGGA ACCGCCA AAGTCCAGGTCTAGATCTAGAGACAGTGCAACAGATCATCATATGGAAGTTATGAGCAGA GAAAAGA CTCTTGAGTTGGCAGCTTCTCAGCCAAGCTCCAAGACGCCGCAAAAGAACCCTTCCATCT ATGCTTG CTCGCTCTGCTCCAAGAGATTCACAAGACCATATAATTTGAAGTCTCACCTTCGCACGCA CGCTGAT GGAAGGCCTTTCCAGTGTTCAATATGCGGGAAGGCATTTGCTCGTTCTCACGACAGAAAG CGTCATG AGGATCTGCATAGTGGTGAACGAAAGTATTGCTGCAAAGGTGTTTTGTCTGACGGAGTAA CTACATG GGGCTGTGAAAAAAGATTTGCCCGAACAGATGCGCTGGGTAGACATTTCAAAACTGAATG TGGTAAA CTGTGTATCAAGCCGCTGATGGATGAACTAAAGAGGGAGGAAGCTTACAGGAGGAATGAA CCAGTAA CAGAAATGAATGACGAGCTTTACTCCCAATCTGTCCAAGATATATTTAGCTCTCAGCGGC TTGGTCA GAACATAGATGACtga

(SEQ ID NO: 2 comprising a1208g mutation)

7. Pichia pastoris mutant CRZ1 isolated from yGLY17159 (encodinq the mutation: F406->S, mutated nucleotide is in bold font) ATGGCAGACCAACGGCTTGAGGATGAGTTTGATATCTCCAGATACTTATCTATTTCTCCT ATCGAGT CAGCTTCAATCGAAGAATCAATCAACGGTTTAATGAGTAGTTGGATTCCTCCGGCTAAGG GTGAGAT TAGAGATTCACTTCCTCCAAACGCTTCTTTTGAAGCTACAGACAGTTTTTCAACCAGTTC ATACCAG GAAATTATACCAGCACAGGTGAAAATAAAACTGGAGTTTGATAATGACCAGCAGCCTGTT TTCTATC AAGAATCGCAACCAGTTTATGATAAGCATTTAACCGTCAATGATCAGGAAACCAGAAGCG CCCAAGA CTTCAACCAATACTTGAATGCTGATGCCGTATCGAGGACCAACTCCATCTCCAACTTATC GGAGCTG TCAACTCATTCCCATATTACCCCTCCAACGCTACTTCATGATCAAGCCTCATTGTCTCCT GCTCTCT TATCTATGAACAGTGATGAAAGAAACGAACTCAATCTGGAAACACTACAGCTAGATCAAA CGTCACA GCCTTACGTGAATCAGATAAAAACGGAGGCAGCTTACGAAGAGCTTTCAGAGTTACACCA CAGATTA GAAAGACTCACTGAGACAAATTTAATTCATCAAGACCAGCTTCAACTCGAACAACAAGAG CAACAAA ATCAGACTCCTCATACTCTCAGTCCTCCTATACAACTTCAGACTCCCATAATCAAAGTCT TGCAAGC CCCAAATGATATAGCAGCAAATACCCCGTCTCTTTTTTCTCAATCTAACCATTCATCTCC ATATAAC ACACCCAAACATTCCAGGTCAAACTCGTTGAGTTCAAA GACAGACAACATGATATTCCACAAATAT CCTCAGTTTTAGACACGTCTTCGTTTTTGGTACCTGGAGATCAGTTTCAAGCAATGAGAG AAGGTAG ACAGAGGAGGAAATCCGAGTCCAACTCTAGAAACTCGAAAGAACGTTCTAAATCTAGGGA ACCGCCA AAGTCCAGGTCTAGATCTAGAGACAGTGCAACAGATCATCATATGGAAGTTATGAGCAGA GAAAAGA CTCTTGAGTTGGCAGCTTCTCAGCCAAGCTCCAAGACGCCGCAAAAGAACCCTTCCATCT ATGCTTG CTCGCTCTGCTCCAAGAGATTCACAAGACCATATAATTTGAAGTCTCACCTTCGCACGCA CGCTGAT GAAAGGCCTTCCCAGTGTTCAATATGCGGGAAGGCATTTGCTCGTTCTCACGACAGAAAG CGTCATG AGGATCTGCATAGTGGTGAACGAAAGTATTGCTGCAAAGGTGTTTTGTCTGACGGAGTAA CTACATG GGGCTGTGAAAAAAGATTTGCCCGAACAGATGCGCTGGGTAGACATTTCAAAACTGAATG TGGTAAA CTGTGTATCAAGCCGCTGATGGATGAACTAAAGAGGGAGGAAGCTTACAGGAGGAATGAA CCAGTAA CAGAAATGAATGACGAGCTTTACTCCCAATCTGTCCAAGATATATTTAGCTCTCAGCGGC TTGGTCA GAACATAGATGACtga

(SEQ ID NO: 2 comprising t1217c mutation)

8. Pichia pastoris mutant CRZ1 isolated from yGLY28999 (encoding the mutation: F406->L, mutated nucleotide is in bold font)

ATGGCAGACCAACGGCTTGAGGATGAGTTTGATATCTCCAGATACTTATCTATTTCTCCT ATCGAGT CAGCTTCAATCGAAGAATCAATCAACGGTTTAATGAGTAGTTGGATTCCTCCGGCTAAGG GTGAGAT TAGAGATTCACTTCCTCCAAACGCTTCTTTTGAAGCTACAGACAGTTTTTCAACCAGTTC ATACCAG GAAATTATACCAGCACAGGTGAAAATAAAACTGGAGTTTGATAATGACCAGCAGCCTGTT TTCTATC AAGAATCGCAACCAGTTTATGATAAGCATTTAACCGTCAATGATCAGGAAACCAGAAGCG CCCAAGA CTTCAACCAATACTTGAATGCTGATGCCGTATCGAGGACCAACTCCATCTCCAACTTATC GGAGCTG TCAACTCATTCCCATATTACCCCTCCAACGCTACTTCATGATCAAGCCTCATTGTCTCCT GCTCTCT TATCTATGAACAGTGATGAAAGAAACGAACTCAATCTGGAAACACTACAGCTAGATCAAA CGTCACA GCCTTACGTGAATCAGATAAAAACGGAGGCAGCTTACGAAGAGCTTTCAGAGTTACACCA CAGATTA GAAAGACTCACTGAGACAAATTTAATTCATCAAGACCAGCTTCAACTCGAACAACAAGAG CAACAAA ATCAGACTCCTCATACTCTCAGTCCTCCTATACAACTTCAGACTCCCATAATCAAAGTCT TGCAAGC CCCAAATGATATAGCAGCAAATACCCCGTCTCTTTTTTCTCAATCTAACCATTCATCTCC ATATAAC ACACCCAAACATTCCAGGTCAAACTCGTTGAGTTCAAA GACAGACAACATGATATTCCACAAATAT CCTCAGTTTTAGACACGTCTTCGTTTTTGGTACCTGGAGATCAGTTTCAAGCAATGAGAG AAGGTAG ACAGAGGAGGAAATCCGAGTCCAACTCTAGAAACTCGAAAGAACGTTCTAAATCTAGGGA ACCGCCA AAGTCCAGGTCTAGATCTAGAGACAGTGCAACAGATCATCATATGGAAGTTATGAGCAGA GAAAAGA CTCTTGAGTTGGCAGCTTCTCAGCCAAGCTCCAAGACGCCGCAAAAGAACCCTTCCATCT ATGCTTG CTCGCTCTGCTCCAAGAGATTCACAAGACCATATAATTTGAAGTCTCACCTTCGCACGCA CGCTGAT GAAAGGCCTCTCCAGTGTTCAATATGCGGGAAGGCATTTGCTCGTTCTCACGACAGAAAG CGTCATG AGGATCTGCATAGTGGTGAACGAAAGTATTGCTGCAAAGGTGTTTTGTCTGACGGAGTAA CTACATG GGGCTGTGAAAAAAGATTTGCCCGAACAGATGCGCTGGGTAGACATTTCAAAACTGAATG TGGTAAA CTGTGTATCAAGCCGCTGATGGATGAACTAAAGAGGGAGGAAGCTTACAGGAGGAATGAA CCAGTAA CAGAAATGAATGACGAGCTTTACTCCCAATCTGTCCAAGATATATTTAGCTCTCAGCGGC TTGGTCA GAACATAGATGACtga

(SEQ ID NO: 2 comprising t1216c mutation)

9. Pichia pastoris mutant CRZ1 isolated from vGLY29030 (encoding the mutation: C41 1 ->F, mutated nucleotide is in bold font)

ATGGCAGACCAACGGCTTGAGGATGAGTTTGATATCTCCAGATACTTATCTATTTCTCCT ATCGAGT CAGCTTCAATCGAAGAATCAATCAACGGTTTAATGAGTAGTTGGATTCCTCCGGCTAAGG GTGAGAT TAGAGATTCACTTCCTCCAAACGCTTCTTTTGAAGCTACAGACAGTTTTTCAACCAGTTC ATACCAG GAAATTATACCAGCACAGGTGAAAATAAAACTGGAGTTTGATAATGACCAGCAGCCTGTT TTCTATC AAGAATCGCAACCAGTTTATGATAAGCATTTAACCGTCAATGATCAGGAAACCAGAAGCG CCCAAGA CTTCAACCAATACTTGAATGCTGATGCCGTATCGAGGACCAACTCCATCTCCAACTTATC GGAGCTG TCAACTCATTCCCATATTACCCCTCCAACGCTACTTCATGATCAAGCCTCATTGTCTCCT GCTCTCT TATCTATGAACAGTGATGAAAGAAACGAACTCAATCTGGAAACACTACAGCTAGATCAAA CGTCACA GCCTTACGTGAATCAGATAAAAACGGAGGCAGCTTACGAAGAGCTTTCAGAGTTACACCA CAGATTA GAAAGACTCACTGAGACAAATTTAATTCATCAAGACCAGCTTCAACTCGAACAACAAGAG CAACAAA ATCAGACTCCTCATACTCTCAGTCCTCCTATACAACTTCAGACTCCCATAATCAAAGTCT TGCAAGC CCCAAATGATATAGCAGCAAATACCCCGTCTCTTTTTTCTCAATCTAACCATTCATCTCC ATATAAC ACACCCAAACATTCCAGGTCAAACTCGTTGAGTTCAAATGACAGACAACATGATATTCCA CAAATAT CCTCAGTTTTAGACACGTCTTCGTTTTTGGTACCTGGAGATCAGTTTCAAGCAATGAGAG AAGGTAG ACAGAGGAGGAAATCCGAGTCCAACTCTAGAAACTCGAAAGAACGTTCTAAATCTAGGGA ACCGCCA AAGTCCAGGTCTAGATCTAGAGACAGTGCAACAGATCATCATATGGAAGTTATGAGCAGA GAAAAGA CTCTTGAGTTGGCAGCTTCTCAGCCAAGCTCCAAGACGCCGCAAAAGAACCCTTCCATCT ATGCTTG CTCGCTCTGCTCCAAGAGATTCACAAGACCATATAATTTGAAGTCTCACCTTCGCACGCA CGCTGAT GAAAGGCCTTTCCAGTGTTCAATATTCGGGAAGGCATTTGCTCGTTCTCACGACAGAAAG CGTCATG AGGATCTGCATAGTGGTGAACGAAAGTATTGCTGCAAAGGTGTTTTGTCTGACGGAGTAA CTACATG GGGCTGTGAAAAAAGATTTGCCCGAACAGATGCGCTGGGTAGACATTTCAAAACTGAATG TGGTAAA CTGTGTATCAAGCCGCTGATGGATGAACTAAAGAGGGAGGAAGCTTACAGGAGGAATGAA CCAGTAA CAGAAATGAATGACGAGCTTTACTCCCAATCTGTCCAAGATATATTTAGCTCTCAGCGGC TTGGTCA GAACATAGATGACtga

(SEQ ID NO: 2 comprising g1232t mutation)

10. Pichia pastoris mutant CRZ1 isolated from vGLY29031 (encoding the mutation:

K469->N, mutated nucleotide is in bold font)

ATGGCAGACCAACGGCTTGAGGATGAGTTTGATATCTCCAGATACTTATCTATTTCTCCT ATCGAGT CAGCTTCAATCGAAGAATCAATCAACGGTTTAATGAGTAGTTGGATTCCTCCGGCTAAGG GTGAGAT TAGAGATTCACTTCCTCCAAACGCTTCTTTTGAAGCTACAGACAGTTTTTCAACCAGTTC ATACCAG GAAATTATACCAGCACAGGTGAAAATAAAACTGGAGTTTGATAATGACCAGCAGCCTGTT TTCTATC AAGAATCGCAACCAGTTTATGATAAGCATTTAACCGTCAATGATCAGGAAACCAGAAGCG CCCAAGA CTTCAACCAATACTTGAATGCTGATGCCGTATCGAGGACCAACTCCATCTCCAACTTATC GGAGCTG TCAACTCATTCCCATATTACCCCTCCAACGCTACTTCATGATCAAGCCTCATTGTCTCCT GCTCTCT TATCTATGAACAGTGATGAAAGAAACGAACTCAATCTGGAAACACTACAGCTAGATCAAA CGTCACA GCCTTACGTGAATCAGATAAAAACGGAGGCAGCTTACGAAGAGCTTTCAGAGTTACACCA CAGATTA GAAAGACTCACTGAGACAAATTTAATTCATCAAGACCAGCTTCAACTCGAACAACAAGAG CAACAAA ATCAGACTCCTCATACTCTCAGTCCTCCTATACAACTTCAGACTCCCATAATCAAAGTCT TGCAAGC CCCAAATGATATAGCAGCAAATACCCCGTCTCTTTTTTCTCAATCTAACCATTCATCTCC ATATAAC ACACCCAAACATTCCAGGTCAAACTCGTTGAGTTCAAATGACAGACAACATGA ATTCCACAAA AT CCTCAGTTTTAGACACGTCTTCGTTTTTGGTACCTGGAGATCAGTTTCAAGCAATGAGAG AAGGTAG ACAGAGGAGGAAATCCGAGTCCAACTCTAGAAACTCGAAAGAACGTTCTAAATCTAGGGA ACCGCCA AAGTCCAGGTCTAGATCTAGAGACAGTGCAACAGATCATCATATGGAAGTTATGAGCAGA GAAAAGA CTCTTGAGTTGGCAGCTTCTCAGCCAAGCTCCAAGACGCCGCAAAAGAACCCTTCCATCT ATGCTTG CTCGCTCTGCTCCAAGAGATTCACAAGACCATATAATTTGAAGTCTCACCTTCGCACGCA CGCTGAT GAAAGGCCTTTCCAGTGTTCAATATGCGGGAAGGCATTTGCTCGTTCTCACGACAGAAAG CGTCATG AGGATCTGCATAGTGGTGAACGAAAGTATTGCTGCAAAGGTGTTTTGTCTGACGGAGTAA CTACATG GGGCTGTGAAAAAAGATTTGCCCGAACAGATGCGCTGGGTAGACATTTCAAAACTGAATG TGGTAAC CTGTGTATCAAGCCGCTGATGGATGAACTAAAGAGGGAGGAAGCTTACAGGAGGAATGAA CCAGTAA CAGAAATGAATGACGAGCTTTACTCCCAATCTGTCCAAGATATATTTAGCTCTCAGCGGC TTGGTCA GAACATAGATGACtga (SEQ ID NO: 2 comprising a1407c mutation)

EXAMPLE 2: Confirmation of Phenotype by Directed Strain Engineering

As discussed herein, independent mutations in the same CRZ1 gene in each of the mutants strongly indicated that inactivation of this transcription factor is responsible for the observed temperature-resistance and fermentation robustness phenotypes. To confirm this conclusion, the CRZ1 ORF was either completely deleted, or the endogenous CRZ1 gene was replaced with the truncated versions shown in Figure 8, in YGLY21203, which is a non- mutagenized ura5 auxtroph Pichia strain derived from YGLY17108 by 5-FOA counter- selection.

Plasmid pGLY12829 (Figure 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 bp of the CRZ1 ORF plus 1 .7 kb of the downstream region. After Sfil digestion, this CRZ7-upstream-URAblaster-CRZ7-downstream DNA fragment was transformed into a non-mutagenized host strain (e.g., YGLY17108). By homologous recombination at both the CRZ1 upstream and downstream regions, this URAblaster- cassette replaced the endogenous CRZ1 gene, deleting 85% of CRZTs coding region, thus generating a complete CRZ1 knock-out mutant. To confirm the correct replacement of the CRZ1 ORF, genomic DNA polymerase chain reaction (PCR) assays were conducted using the following oligonucleotides as PCR primers: GTATGCGATATAGTGTGGA (SEQ ID NO: 4, 1545 bp 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 bp downstream of the CRZ1 stop codon) to confirm the 3' junction of the gene-replacement; plus "

GACACATGCGAAATGTCCTG " (SEQ ID NO:8, 120 bp downstream of the CRZ1 stop codon) and "TTGAGTTGGCAGCTTCTCAG" (SEQ ID NO: 9, within the CRZ1 ORF, 1075 bp after the start) to confirm the absence of the wild-type CRZ1 ORF. Plasmid pGLY12832 (Figure 7) was constructed by cloning a 1.5 kb DNA fragment (0.6 kb upstream region, the 1st 879 bp 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 bp of the CRZ1 ORF plus 1.7 kb of the downstream region. Homologous recombination-mediated double-crossovers between the Sfil-fragment of pGLY12832 (Figure 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. After confirming that the DNA constructs precisely replaced the endogenous CRZ1 gene with the corresponding deletion or truncations, their abilities to grow at 35°C on solid media and 32°C in liquid media in a bioreactor was examined. It was confirmed that these CRZ1 truncation and deletion mutants displayed temperature-resistant phenotypes very similar to those observed from the original mutants isolated by UV mutagenesis.

Next, the truncation and deletion mutants were subjected to standard DasGip MeOH fed-batch fermentation runs (Hopkins et a/., 201 1 ) to determine whether they would also display increased fermentation robustness at 32°C. As shown in Figure 9, the YGLY17108 control strain displayed heavy lysis and was not viable within 65 hours of MeOH induction at 32°C. In contrast, 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. These results demonstrated that CRZ1 in-activation, by deleting the CRZ1 ORF completely or partially, is sufficient for improving the fermentation robustness of glyco-engineered strains. Furthermore, 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.

Inactivation of the ATT1 gene has resulted in a dramatic improvement in strain fermentation robustness. Because the inactivation of CRZ1 and ATT1 gave rise to very similar phenotype (i.e., temperature-resistance and enhanced fermentation robustness), we want to examine if CRZ1 deletion would further improve the fermentation robustness of a strain already containing an ATT1 deletion mutation. To this end, we constructed crzl, attl double deletion mutants and tested their fermentation robustness by carrying out standard MeOH fed-batch fermentation runs in 1 L DasGip bioreactors at 34°C. Under this very stringent fermentation condition, the attl single deletion strain YGLY29128 remained viable for 75 hours, whereas the crzl, attl double mutants remained viable for more than 1 15 hours (Figure 10). These results clearly demonstrated that the fermentation robustness improvements derived from attl deletion and crzl deletion are additive, and that crzl, attl double mutants displayed much higher level of fermentation robustness than the single mutants.

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The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, the scope of the present invention includes embodiments specifically set forth herein and other embodiments not specifically set forth herein; the embodiments specifically set forth herein are not necessarily intended to be exhaustive.

Various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the claims.

Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.