WO2012170292A1

WO2012170292A1 - Modulation of metabolic pathways for improving bioprocess performance and secreted protein productivity of yeast

Info

Publication number: WO2012170292A1
Application number: PCT/US2012/040393
Authority: WO
Inventors: Robert Davidson
Original assignee: Merck Sharp & Dohme Corp.
Priority date: 2011-06-07
Filing date: 2012-06-01
Publication date: 2012-12-13

Abstract

An isolated fungal host cell, such as Pichia pastoris, lacking full wild-type levels of dihydroxyacetone synthase activity due to a knocked- out DAS1 or DAS2, is provided. The host cell further comprises a heterologous polynucleotide encoding a heterologous polypeptide. Methods for expressing the heterologous polypeptide in such cells, and purifying the expressed polypeptide are also disclosed.

Description

MODULATION OF METABOLIC PATHWAYS FOR IMPROVING

BIOPROCESS PERFORMANCE AND SECRETED PROTEIN

PRODUCTIVITY OF YEAST

This Application claims the benefit of U.S. Provisional Patent

Application No. 61/494,084, filed June 7, 2011; which is herein incorporated by reference in its entirety.

Field of the Invention

The present invention relates to isolated fungal cells such as

Pichia pastoris comprising a heterologous polypeptide and a knocked-out DASl or DAS2 as well as method of making and using the same . Background of the Invention

The methylotrophic yeast Pichia pastoris is a widely utilized expression host for the production of heterologous proteins . As a single-celled microbe with well -developed genetic tools, P.

pastoris provides the ability for rapid and extensive genetic engineering, leading to shorter cycle times and improved product quality attributes. Furthermore, the capability of achieving high fermentation cell densities combined with the notably strong methanol -inducible AOX1 promoter results in secreted protein productivities reported in the multi-gram per liter range.

Importantly, P. pastoris is a eukaryote, and as such contains basic machinery for protein folding and post-translational modifications.

After methanol passively diffuses into the Pichia pastoris peroxisome, it is converted to formaldehyde by one of two different alcohol oxidase isozymes (AOX1, AOX2) . Following this, one of two pathways can be utilized. Formaldehyde can be further oxidized in several steps to C0₂ via the methanol dissimilatory pathway. This pathway also yields two NADH reducing equivalents that are used for energy generation. Alternatively, formaldehyde can be incorporated into the pentose phosphate pathway via a condensation reaction with xylulose 5 -phosphate, a reaction catalyzed by a specialized transketolase enzyme called DiHydroxyAcetone Synthase (DHAS or DAS) . This reaction yields a molecule of dihydroxyacetone (DHA) and a molecule of glyceraldehyde 3 -phosphate (G3P) . Each of these reactions occurs in peroxisomes in Pichia pastoris and other methylotrophic yeasts, and this compartmentalization is critical to the efficient use of methanol as a carbon source. At this point in the pathway, these two C3 molecules (DHA and G3P) can exit the peroxisome, and following phosphorylation of DHA by

dihydroxyacetone kinase (DAK) , enter glycolysis or gluconeogenesis to generate energy and maintain the myriad of other central carbon pathways. Each iteration of this cycle incorporates a single molecule of formaldehyde from methanol into the central carbon network. This accounts for the abundance of the enzymes involved (Aoxl/2p can be up to 30% of the cellular protein levels) but also why the pathway is under such tight regulation. These properties result in a promoter (P_aoxi) , that is one of the most strongly inducible, to be identified, but also a protein production that is complex to optimize and often difficult to scale. However, through a better understanding of methanol metabolism and the impact of process variables on protein secretion, significant improvements can be made in productivities.

Most of the genes in the methanol pathway have been

identified. In addition to the genes encoding the Aoxl/2p

isozymes, a gene encoding DAS, and many of the peroxisome enzymes have been identified. The peroxisomal catalase gene, which is required to cope with the massive amounts of H₂0₂ generated by

Aoxl/2 has also been cloned and studied. The FLD1 gene, encoding the first enzyme of the aforementioned methanol dissimilatory pathway, has been identifed and the promoter used as an alternative to AOXl due to its unique property of high level induction on methylamine as a sole nitrogen source, in addition to methanol induction.

Despite the complexity of regulation, process drawbacks involved in cultivation on methanol, and the identification of other promoter systems such as GAPDH, most researchers have relied upon the AOXl promoter for production of heterologous proteins . Much research has focused on optimizing the transcriptional potential of this system. The MXR1 gene has been identified as a key positive acting transcription factor in the pathway. Mutation analysis of P_a0xi and also P_a0x2 have led to modified versions of these promoters with unique properties, including increased levels of derepression upon starvation or reduced carbon catabolite repression levels. However, the final process impact in terms of yield from these promoter modifications has been quite modest in most cases. The regulation of these promoters is quite complicated and dependent on significant process optimization to capture any small scale improvements. But, more importantly, it has not been demonstrated that the secretion of heterologous proteins is limited by transcription in this production system. In certain cases it has been shown that additional copies of a gene expression cassette can even have a negative impact on productivity. This is possibly due to a number of other factors including protein synthesis, secretion and folding bottlenecks, as well as gene silencing or transcription factor quenching but it would at least suggest that transcription is not a major bottleneck to higher process yields. More recent work has suggested that such bottlenecks can be at least partially alleviated by genetic intervention in the form of chaperone expression.

Recent progress in the field, including humanization of the P. pastoris N-glycosylation pathway and a better understanding of the yeast secretory pathway, has resulted in improvements in the ability to produce mammalian proteins including monoclonal

antibodies (mAbs) . However, despite these advantages, expression of heterologous proteins has yielded varying results with

efficiencies ranging from several mg/L to several g/L of secreted purified product. Expression of certain heterologous proteins has been shown to be toxic to the host, and combined with the toxicity of methanol itself, can affect the viability of the culture. This, in turn, can affect the quality of the product through the activity of proteases, either secreted or resulting from excessive cell death. Moreover, fermentation processes based on methanol as a sole carbon source have the disadvantages of generating significant amounts of heat and an enormous oxygen requirement, which also can affect cell viability. Attempts to address these issues have ranged from modifying culture conditions, such as dissolved oxygen, temperature, and pH, to optimizing specific growth rate through controlled methanol feeding.

Here, mutant strains are constructed with defects in methanol assimilation. For example, DASl is the first enzyme in the assimilation of methanol via formaldehyde. A dasl mutant strain allows reduced flux through the methanol assimilatory pathway, while shunting flux to the dissimilatory pathway, and thus more controlled growth without significantly reducing (and potentially increasing) energy stores in the form of NADH and ATP.

Summary of the Invention

The present invention provides, in part, an isolated fungal host cell {e.g. , a yeast such as Pichia, e.g., Pichia pastoris) lacking full wild-type levels of dihydroxyacetone synthase

activity, for example, due to a knocked-out DASl or DAS2, which host cell comprises a heterologous polynucleotide (e.g., operably linked to a promoter, e.g., in a vector) encoding an heterologous polypeptide such as, for example, an immunoglobulin heavy and/or light chain. In an embodiment of the invention, the DASl or DAS2 is knocked out by interrupting, point mutating or deleting

chromosomal DASl or DAS2 in the fungal cell; for example, wherein the fungal cell is Pichia pastoris and DASl is knocked-out by interrupting chromosomal DASl by causing genetic recombination of a Pichia pastoris URA5 gene at the DASl chromosomal locus as

described in Figure 4. Optionally, such a fungal host cell

overexpresses one or more members of the cellular methanol

dissimilation pathway, e.g., FLD1, FGH1 or FADl. A growth medium, e.g., a. liquid growth medium, comprising such a fungal host cell also forms part of the present invention. In an embodiment of the invention, the fungal host cell is genetically modified to cause an altered glycosylation pattern on heterologous polypeptide it expresses. In an embodiment of the invention, in the fungal host cell of the present invention: (i) the heterologous polynucleotide encodes a heterologous polypeptide which is an immunoglobulin wherein said immunoglobulin forms part of an antibody or antigen-binding fragment thereof that bind specifically to VEGF, HERl, HER2, HER3, glycoprotein Ilb/lIIa, CD52, IL-2R alpha receptor (CD25) , epidermal growth factor receptor (EGFR) , Complement system protein C5, CDlla, TNF alpha, CD33, IGF1R, CD20, T cell CD3 Receptor, alpha-4 (alpha 4) integrin, PCSK9, immunoglobulin E (IgE) , RSV F protein or ErbB2. Other examples of said heterologous polynucleotides encode: VEGF, HERl, HER2, HER3, glycoprotein Ilb/lIIa, CD52, IL-2R alpha receptor

(CD25) , epidermal growth factor receptor (EGFR) , Complement system protein C5, CDlla, TNF alpha, CD33, IGF1R, CD20, T cell CD3

Receptor, alpha-4 integrin, PCSK9, immunoglobulin E (IgE) , RSV F protein or ErbB2; or wherein said immunoglobulin forms part of an antibody or antigen-binding fragment thereof tht is Abciximab;

Adalimumab; Alemtuzumab; Basiliximab; Bevacizumab; Cetuximab;

Certolizumab; Daclizumab; Dalotuzumab; Denosumab; Eculizumab;

Efalizumab; Gemtuzumab; Ibritumomab tiuxetan; Infliximab;

Muromonab-CD3 ; Natalizumab; Omalizumab; Palivizumab; Panitumumab; Ranibizumab; Rituximab; Tositumomab; or Trastuzumab.

(ii) the DAS1 polynucleotide comprises a nucleotide sequence that is at least 80% (e.g. , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identical to the nucleotide sequence set forth in SEQ ID NO: 2 ;

(iii) the DAS2 polynucleotide comprises a nucleotide sequence that is at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identical to the nucleotide sequence set forth in SEQ ID NO: 3;

(iv) the DAS1 polypeptide comprises an amino acid sequence that is at least 80% (e.g. , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,

98%, 99%, 100%) identical or similar to the amino acid sequence set forth in SEQ ID NO: 4;

(v) the DAS2 polypeptide comprises an amino acid sequence that is at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identical or similar to the amino acid sequence set forth in SEQ ID NO: 5; (vi) the heterologous polynucleotide is operably linked to a methanol-indueiable promoter; or

(vii) said cell produces glycoproteins comprising

GlcNAc₂Man₃GlcNAc₂.

The present invention further provides a method for producing such an isolated fungal host cell which comprises a heterologous polynucleotide encoding a heterologous polypeptide {e.g., an immunoglobulin heavy and/or light chain) comprising knocking out one or more dihydroxyacetone synthases, e.g. , chromosomal DASl or DAS2, in a fungal cell and introducing said polynucleotide into the cell.

The present invention also provides a method for producing a heterologous polypeptide (e.g., an immunoglobulin heavy and/or light chain) comprising introducing a heterologous polynucleotide encoding said polypeptide into an isolated P. pastoris cell in which DASl or DAS2 is genetically knocked-out and culturing said cell under conditions where the polypeptide is expressed in said cell. For example, in an embodiment of the invention, the

heterologous polypeptide is secreted from the host cell and/or the polypeptide is purified after it is expressed by the host cell.

Brief Description of the Figures

Figure 1: Methanol utilization pathway of P. pastoris. The central pathways involved in the metabolism of methanol are depicted. AOXl/2 : Alcohol Oxidase (EC 1.1.3.13), CAT (EC

1.11.1.6): Catalase, DAS (EC 2.2.1.3): Dihydroxyacetone Synthase, DAK (EC 2.7.1.29): Dihydroxyacetone Kinase, TPI (EC 5.3.1.1):

Triose Phosphate Isomerase, FBA (EC 4.1.2.13): Fructose 1,6- bisphosphate Aldolase, FBP (EC 3.1.3.11): Fructose 1,6- bisphosphatase, TAL (EC 2.2.1.2): Transaldolase, TKT (EC 2.2.1.1): Transketolase, RKI (EC 5.3.1.6): Ribulose 5 -phosphate ketol- isomerase, RPE (EC 5.1.3.1): Ribulose phosphate 3-epimerase,

FLD/DH1 (EC 1.1.1.284): S- (hydroxymethyl ) glutathione dehydrogenase, DYH (EC 3.1.2.12): S- formylglutathione hydrolase, FAD (EC 1.2.1.2): Formate Dehydrogenase. Genes encoding these activities in P.

pastoris are described in Table 1. eOH: methanol, Fmd: formaldehyde, For: formate, DHA: dihydroxyacetone, DHAP: dihydroxyacetone phosphate, G3P: glyceraldehyde 3 -phosphate,

F1,6BP: fructose 1 , 6-bisphosphate , F6P: fructose 6 -phosphate, E4P: Erythrose 4 -phosphate, S7P: Septehepulose 7 -phosphate, X5P:

Xylulose 5 -phosphate, R5P, Ribulose 5 -phosphate, Rib5P: Ribose 5- phosphate .

Figure 2: Intensity cluster of gene expression ratios for genes in the methanol pathway. The genes encoding the metabolic machinery depicted in Fig 1 were plotted against the wild type

(yll430) and a glycoengineered Pichia strain (YGLY8323) cultivated on glycerol and methanol. Intensity profiles were ratioed to glycerol samples and are the result of four individual replicates. Genes are named by systematic gene ID and common name from the yll430 strain genome sequence. The systematic gene IDs

corresponding to the GS115 genome sequence are shown in Table 1. Black indicates upregulated and white indicates downregulated with saturation as indicated in the legend. Figure 3: Comparative pathway flux for wild type vs. dasl mutant strains. Deletion of dasl will reduce the potential flux through the assimilation pathway and result in excess formaldehyde being processed by the dissimilation pathway. This results in increased formation of C0₂ but also a higher energy :biomass ratio per unit methanol, which is favorable for protein synthesis, folding, and secretion. MeOH: methanol, Fmd: formaldehyde, ΆΟΧ1/2, FLD1, and DASl/2 genes are described in Table 1.

Figure 4: Deletion of P. pastoris DASl gene. The region of Chromosome 3 where the DASl and DAS2 genes are located is depicted along with the dasl::URA5 allele, which is integrated to generate a dasl mutant strain. The dasl mutant strains are still competent for growth on methanol as a sole carbon source due to the activity of the DAS2 gene, but the capacity to assimilate methanol into biomass is reduced. Figure 5: Restriction map of plasmid pGLY9903 containing the dasl: :URA5 mutant allele. The E. coli/P. pastoris shuttle vector is depicted circularly as it is maintained in E. coli. For introduction into P. pastoris the plasmid is digested with Sfil to release the pUC19 portion, directing integration to the DAS1 locus and replacing the Open Reading Frame of DAS1 with that of the P. pastoris LacZ-URA5-LacZ blaster gene (Nett, 2003) . The GAPDH promoter and CYC1 transcriptional terminator (TT) flank Notl/Pacl sites that can be used for introducing heterologously expressed open reading frames. Locations of restriction enzyme recognition sites are indicated.

Figure 6: Monoclonal antibody productivity of dasl mutant strains. The DAS1 gene was deleted in a P. pastoris strain

engineered to produce human N-glycans and expressing the H chain and L chain genes for an anti-HER2 mAb under control of the AOX1 promoter. Two dasl mutant strains were cultivated in 5ml micro24 (Applikon) mini bioreactors and induced with methanol for 72 hours and the resulting supernatant purified by protein A and analyzed for mAb titer by HPLC. Data are plotted in mg/L of total broth volume (adjusted for cell volume) .

Figure 7 : Robustness of dasl mutant strains under bioreactor conditions. The supernatants from the fermentations described in Figure 6 were subjected to Picogreen assays to measure supernatant concentration of double stranded DNA using a fluorescence-based method. Data are plotted as mcg/ml .

Figure 8: Restriction map of plasmid pGLY-MeOHD!S containing the FLD1, FGH1, and FMDH gene expression cassettes. The E. coli/P. pastoris shuttle vector is depicted circularly as it is maintained in E. coli. For introduction into P. pastoris the plasmid is digested with Spel to linearize the plasmid, directing integration to the URA6 locus via single crossover roll-in. The PpAOXl promoter and transcriptional terminator (TT) cassettes each flank EcoRl/Fsel sites into which the FLD1, FGH1 and FMDH open reading frames have been inserted. Locations of restriction enzyme recognition sites are indicated.

Figure 9: Restriction map of plasmid pGLY-FLDl containing the PpFLDl expression cassette. The E. coli/P. pastoris shuttle vector is depicted circularly as it is maintained in E. coll. For introduction into P. pastoris the plasmid is digested with Spel to linearize the plasmid, directing integration to the URA6 locus via single crossover roll -in. The PpAOXl promoter and transcriptional terminator (TT) cassette flanks EcoRl/Fsel sites into which the FLD1 open reading frame has been inserted. Locations of

restriction enzyme recognition sites are indicated.

Figure 10: Restriction map of plasmid pGLY-FGHl containing the PpFGHl expression cassette. The E. coli/P. pastoris shuttle vector is depicted circularly as it is maintained in E. coli. For introduction into P. pastoris the plasmid is digested with Spel to linearize the plasmid, directing integration to the URA6 locus via single crossover roll -in. The PpAOXl promoter and transcriptional terminator (TT) cassette flanks EcoRl/Fsel sites into which the FGH1 open reading frame has been inserted. Locations of

restriction enzyme recognition sites are indicated.

Figure 11: Restriction map of plasmid pGLY-F DH containing the PpFMDH expression cassette. The E. coli/P. pastoris shuttle vector is depicted circularly as it is maintained in E. coli. For introduction into P. pastoris the plasmid is digested with Spel to linearize the plasmid, directing integration to the URA6 locus via single crossover roll -in. The PpAOXl promoter and transcriptional terminator (TT) cassette flanks EcoRl/Fsel sites into which the FMDH open reading frame has been inserted. Locations of

restriction enzyme recognition sites are indicated.

Detailed Description of the Invention

The present invention encompasses fungal cells, e.g., Pichia cells, that exhibit increased metabolism of formaldehyde in the dissimilation pathway (as shown in figure 1) as a result of lack of full dihydroxyacetone synthase activity e.g., due to knock out of one or more dihydroxyacetone synthases, for example, DAS1 or DAS2. Such cells exhibit superior production of heterologous proteins that have been introduced into the cells, such as immunoglobulins, e.g., during methanol induction of promoters operably linked to polynucleotides encoding such immunoglobulins. Methods for producing such heterologous proteins in such fungal cells are also part of the present invention.

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. Such techniques are explained in the literature. See, e.g., Sambrook, Fritsch &

Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook, et al . , 1989") ; DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985) ; Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985)); Transcription And Translation (B.D. Hames & S.J. Higgins, eds. (1984)); Animal Cell Culture (R.I. Freshney, ed. (1986) ) ; Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F.M. Ausubel, et al . (eds.), Current Protocols in Molecular

Biology, John Wiley & Sons, Inc. (1994) .

Knocking-out a gene on one or both chromosomes, such as DAS1 or DAS2, in a fungal cell of the present invention refers to genetically mutating the chromosomal nucleotide sequence in the cell to cause the cell to express less than normal, wild- type levels of dihydroxyacetone synthase activity, for example, due to decreased levels of expression of the protein or decreased activity of the protein. A knocked-out DAS1 or DAS2 gene may be indicated as "dasl" or "das2". Homozygous knock-outs may be referred to as "das " or "das2^~/'" and heterozygous knock-outs may be referred to as "dasl^+/~" or "das2^+/'" . For example, such mutations can be done by partially or fully deleting, disrupting or point mutating (in one or more places) the gene coding sequence of DASl or DAS2 or by mutating the expression regulator sequences of DASl or DAS2 (e.g., promoter) or by mutating another gene or regulatory sequence thereof that is required for full, normal expression of DASl or DAS2. In an embodiment of the invention, a fungal host cell of the present invention is not doubly dasl das2 knocked-out , but, rather singly knocked out, i.e., dasl DAS2 or DASl das2.

A "heterologous polynucleotide" in a fungal cell of the present invention in which DASl or DAS2 is knocked-out is a polynucleotide that does not naturally occur in the cell, e.g., because the nucleotide sequence of the polynucleotide does not naturally occur in the fungal cell. A "heterologous polypeptide", is a polypeptide that does not naturally occur in the cell, e.g., because the amino acid sequence of the polypeptide does not naturally occur in the fungal cell. An example of a heterologous polynucleotide encoding a heterologous polypeptide is an antibody immunoglobulin heavy chain or light chain.

Examples of an antibody containing an immunoglobulin which can be encoded by a heterologous polynucleotide in a fungal host cell of the present invention, in which DASl or DAS2 is knocked-out, are antibodies that bind specifically to VEGF, HER1, HER2 , HER3 , glycoprotein lib/Ilia, CD52, IL-2R alpha receptor (CD25) , epidermal growth factor receptor (EGFR) , Complement system protein C5, CDlla, TNF alpha, CD33, IGF1R, CD20, T cell CD3 Receptor, alpha-4 (alpha 4) integrin, PCSK9, immunoglobulin E (IgE) , RSV F protein or ErbB2. Other examples of said heterologous polynucleotides encode: VEGF, HER1, HER2, HER3 , glycoprotein Ilb/lIIa, CD52, IL-2R alpha receptor (CD25) , epidermal growth factor receptor (EGFR) , Complement system protein C5, CDlla, TNF alpha, CD33, IGF1R, CD20, T cell CD3

Receptor, alpha-4 (alpha 4) integrin, PCSK9, immunoglobulin E

(IgE), RSV F protein or ErbB2 ; or an immunogenic fragment thereof; or said heterologous polynucleotides encode the light chain or heavy chain immunoglobulin of Abciximab; Adalimumab; Alemtuzumab; Basiliximab; Bevacizumab; Cetuximab; Certolizumab; Daclizumab;

Dalotuzumab; Denosumab; Eculizumab; Efalizumab; Gemtuzumab;

Ibritumomab tiuxetan; Infliximab; uromonab-CD3 ; Natalizumab; Omalizumab; Palivizumab; Panitumumab; Ranibizumab; Rituximab;

Tositumomab; or Trastuzumab.

A "polynucleotide" , "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 .

A "coding sequence" or a sequence "encoding" an expression product, such as an RNA or polypeptide is a nucleotide sequence

(e.gr., 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 ³²P-nucleotides , ³H-nucleotides , ¹⁴C-nucleotides, ³⁵S-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated.

"Overexpression" and the like refers to expression of a protein in a cell at levels greater than normal in a wild-type cell.

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. A polypeptide may be indicated with a "p" at the end of a polypeptide name, for example, Daslp or Das2p would refer to the Dasl

polypeptide and the Das2 polypeptide.

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.

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 al . , 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.

A coding sequence {e.g., of a heterologous polynucleotide, e.g., an 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) 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. In an embodiment of the invention, a

polynucleotide is operably linked to a transcriptional terminator sequence .

The present invention includes fungal host cells, in which DAS1 or DAS2 is knocked-out, having a vector which comprises a promoter operably linked to a heterologous polynucleotide. 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. In general, a plasmid is circular, includes an origin {e.g., 2 origin) and, preferably includes a selectable marker. In plasmids which can be maintained in yeast, commonly used yeast markers include URA3 , HIS3 , LEU2, TRPl and LYS2, which complement specific auxotrophic mutations in a yeast host cell, such as ura3-52, his3-Dl, Ieu2-Dl, trpl-Dl and lys2-201, respectively. If the plasmid can be maintained in

E.coli, it may include a bacterial origin (ori) and/or a selectable market such as the β- lactamase gene (bla or AMP) . Commonly used yeast/E. coll shuttle vectors are the Yip (see Myers et al . , Gene 45: 299-310, (1986)), YEp (see Myers et al., Gene 45: 299-310, (1986)), YCp and YRp plasmids . The Yip integrative vectors do not replicate autonomously, but integrate into the genome at low frequencies by homologous recombination. The YEp yeast episomal plasmid vectors replicate autonomously because of the presence of a segment of the yeast 2 μιτι plasmid that serves as an origin of replication (2 μηι ori) . The 2 μιη ori is responsible for the high copy-number and high frequency of transformation of YEp vectors. The YCp yeast centromere plasmid vectors are autonomously

replicating vectors containing centromere sequences, CEN, and autonomously replicating sequences, ARS. The YCp vectors are typically present at very low copy numbers, from 1 to 3 per cell. Autonomously replicating plasmids (YRp) which carry a yeast origin of replication (ARS sequence; but not centromere) that allows the transformed plasmids to be propagated several hundred- fold. Yip, YEp, YCp and YRp are commonly known in the art and widely

available. Another acceptable yeast vector is a yeast artificial chromosome (YAC) . A yeast artificial chromosome is a biological vector. It is an artificially constructed chromosome and contains the telomeric, centromeric, and replication origin sequences needed for replication in yeast cells (see Marchuk et al . , Nucleic Acids Res. 16(15) : 7743 (1988); Rech et al . , Nucleic Acids Res.

18 (5) : 1313 (1990) ) .

Vectors that could be used in this invention include plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles that may facilitate introduction of the nucleic acids into the genome of a host cell (e.g., Pichia pastoris) . Plasmids are the most commonly used form of vector but all other forms of vectors which serve a similar function and which are, or become, known in the art are suitable for use herein. See, e.g., Pouwels, et al., Cloning Vectors: A Laboratory Manual, 1985 and Supplements, Elsevier, N.Y. , and Rodriguez et al . (eds.), Vectors : A Survey of Molecular Cloning Vectors and Their Uses, 1988, Buttersworth, Boston, MA.

The term methanol -induction and the like refers to increasing expression of a polynucleotide (e.g., a heterologous

polynucleotide) operably linked to a methanol -inducible promoter. AOX1 promoter is an example of a methanol-inducible promoter. The scope of the present invention includes fungal host cells, in which DAS1 or DAS2 is knocked-out, comprising a heterologous

polynucleotide, encoding a heterologous polypeptide, that is operably linked to a promoter, for example, a methanol -inducible promoter, e.g., AOX1, e.g., in a vector. Methods for expressing a heterologous polypeptide in such a fungal host cell are part of the present invention. For example, such a method comprises

introducing such a methanol -inducible promoter-heterologous polynucleotide construct into such a fungal cell and culturing the host cell in the presence of methanol under conditions whereby the polypeptide is expressed.

The present invention includes fungal host cells in which DAS1 or DAS2 is knocked-out, for example, wherein the DAS1 comprises the nucleotide sequence SEQ ID NO: 2 and/or wherein the DAS2 comprises the nucleotide sequence SEQ ID NO: 3. In an embodiment of the invention, the DAS1 and/or DAS2 is a sequence variant thereof which comprises a nucleotide sequence that differs from but also

hybridizes to a complement of a polynucleotide having a nucleotide sequence set forth in SEQ ID NO: 2 or 3. Preferably, the

polynucleotides hybridize under low stringency conditions, more preferably under moderate stringency conditions and most preferably under high stringency conditions. A polynucleotide is

"hybridizable" to another polynucleotide when a single stranded form of the nucleic acid molecule (e.g., either strand) can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook, et al., supra) . The conditions of temperature and ionic strength determine the "stringency" of the hybridization. Low stringency hybridization conditions may be 55°C, 5X SSC, 0.1% SOS, 0.25% milk, and no formamide; or 30% formamide, 5X SSC, 0.5% SDS . Moderate stringency hybridization conditions are similar to the low

stringency conditions except the hybridization is carried out in 40% formamide, with 5X or 6X SSC. High stringency hybridization conditions are similar to low stringency conditions except the hybridization conditions are carried out in 50% formamide, 5X or 6X SSC and, optionally, at a higher temperature (e.g. , 57°C, 59°C, 60°C, 62°C, 63°C, 65°C or 68°C) . In general, SSC is 0.15M NaCl and 0.015M sodium citrate. Hybridization requires that the two nucleic acids contain complementary sequences, although, depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the higher the stringency under which the nucleic acids may hybridize. For hybrids of greater than 100 nucleotides in length, equations for calculating the melting temperature have been derived (see Sambrook, et al., supra, 9.50-9.51) . For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the

oligonucleotide determines its specificity (see Sambrook, et al . , supra, 11.7-11.8) .

In an embodiment of the invention, the DAS1 and/or DAS2 polynucleotide comprises a nucleotide sequence which is at least about 70% identical, preferably at least about 80% identical, more preferably at least about 90% identical and most preferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to SEQ ID NO: 2 or 3 when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences; but which encode a polypeptide that retains dihydroxyacetone synthase activity, e.g., at a detectable level or at a level at least equal to that of the corresponding non-variant Daslp or Das2p. In an embodiment of the invention, the Daslp and/or Das2p polypeptide comprises an amino acid sequence which is at least about 70% similar or identical, preferably at least about 80% similar or identical, more preferably at least about 90% similar or identical and most preferably at least about 95% similar or identical {e.g., 95%, 96%, 97%, 98%, 99%, 100%) to SEQ ID NO: 4 or 5 when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences; but which retains dihydroxyacetone synthase activity, e.g., at a detectable level or at a level at least equal to that of the corresponding non-variant Daslp or Das2p .

Sequence identity refers to exact matches between the

nucleotides or amino acids of two sequences which are being compared. Sequence similarity refers to both exact matches between the amino acids of two polypeptides which are being compared in addition to matches between nonidentical, biochemically related amino acids which may be interchangable .

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; ootton, 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. Host Cells

The present invention encompasses isolated fungal host cells (e.g., Pichia cells such as Pichia pastoris) lacking full

dihydroxyacetone synthase activity, e.g. , due to knock-out of 1 or more dihydroxyacetone synthases such as DAS1 or DAS2, including a polynucleotide encoding a heterologous polypeptide {e.g., an immunoglobulin chain) . For example, in an embodiment of the invention, the heterologous polypeptide is encoded by a

heterologous polynucleotide operably linked to a promoter, e.g., a methanol inducible promoter.

Fungal host cells of the present invention may be genetically engineered so as to express particular glycosylation patterns on polypeptides {e.g. , immunoglobulins) that are expressed in such cells. Fungal host cells of the present invention are discussed in detail herein.

A "fungal host cell" that may be used in a composition or method of the present invention, as is discussed herein, includes cells lacking full dihydroxyacetone synthase activity (e.g. , due to knock out of DAS1 or DAS2) and including a heterologous

polynucleotide encoding a heterologous polypeptide (e.g., an immunoglobulin) . In an embodiment of the invention, the fungal host cell is a yeast cell, such as a methylotrophic yeast cell, which, for example, is selected from the group consisting of any Pichia cell, Pichia pastoris, 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, Pichia methanolica, Pichia, Saccharomyces cerevisiae, Saccharomyces, Hansenula polymorpha, Kluyveromyces, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei ,

Chrysosporium lucknowense, Fusarium, Fusa um gramineum, Fusarium venenatu and Neuraspora crassa.

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 Man₃GlcNAc₂ ("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 Man₃GlcNAc₂ ("Man₃") 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. Hybrid N-glycans may also have a galactose ("Gal") or N-acetylgalactosamine ( "GalNAc" ) residue that are optionally modified with sialic acid or derivatives (e.g.,

"NANA" or "NeuAc") attached to the GlcNAc on the 1,3-mannose arm. The various N-glycans are also referred to as "glycoforms . "

"PNGase", or "glycanase" or "glucosidase" refer to peptide N- glycosidase F (EC 3.2.2.18) .

In an embodiment of the invention, O-glycosylation of

glycoproteins in a fungal host cell is controlled. The scope of the present invention includes isolated fungal host cells (e.g. , Pichia) wherein O-glycosylation is controlled (as discussed herein) and methods of use thereof. For example, fungal host cells are part of the present invention wherein O-glycan occupancy and mannose chain length are reduced. In lower eukaryote host cells such as yeast, O-glycosylation can be controlled by deleting the genes encoding one or more protein O-mannosyltransferases (Dol- PMan: Protein (Ser/Thr) Mannosyl Transferase genes) (PMTs) or by growing the host in a medium containing one or more Pmtp

inhibitors. Thus, the present invention includes isolated fungal host cells lacking full dihydroxyacetone synthase activity (e.g., due to knock out of DAS1 or DAS2) and including a polynucleotide encoding a heterologous polypeptide (e.g., an immunoglobulin chain), e.g., comprising a deletion of one or more of the genes encoding PMTs, and/or, e.g., wherein the host cell can be

cultivated in a medium that includes one or more Pmtp inhibitors. Pmtp inhibitors include but are not limited to a benzylidene thiazolidinedione . Examples of benzylidene thiazolidinediones are 5-[[3,4bis( phenylmethoxy) phenyl] methylene] -4-oxo-2-thioxo-3- thiazolidineacetic Acid; 5- [[3- (1-25 Phenylethoxy) -4- (2- phenylethoxy) ] phenyl] methylene] -4-oxo-2-thioxo-3-thiazolidineacetic Acid; and 5- [ [3- (1 -Phenyl -2 -hydroxy) ethoxy) -4- (2- phenylethoxy) ] phenyl] methylene] -4-oxo-2-thioxo3-thiazolidineacetic acid. In an embodiment of the invention, a fungal host cell (e.g. , Pichia) includes a nucleic acid that encodes an alpha-1,2- mannosidase that has a signal peptide that directs it for

secretion. For example, in an embodiment of the invention, the fungal host cell is engineered to express an exogenous alpha- 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 Man₈GlcNAc₂ to yield Man_sGlcNAc₂. See U.S. Patent no. 7,029,872.

Fungal host cells (e.g., Pichia) 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 beta-mannosyltransferasegenes (e.g., BMTl , BMT2, BMT3, and BMT4) (See, U.S. Published Patent Application No.

2006/0211085) or abrogating translation of RNAs encoding one or more of the beta-mannosyltransferasesusinginterfering RNA,

antisense RNA, or the like. The scope of the present invention includes such isolated fungal host cells (e.g., Pichia) lacking full dihydroxyacetone synthase activity (e.g. , due to knock out of DAS1 or DAS2) and including a polynucleotide encoding a

heterologous polypeptide (e.g., an immunoglobulin chain) .

Fungal host cells (e.g., Pichia) 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 PNOl and MNN4B (See for example, U.S. Patent Nos. 7,198,921 and 7,259,007), which can include deleting or disrupting the MNN4A gene 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, a fungal host cell has been genetically modified to 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 Man₃GlcNAc₂, GlcNAC₍i- ₄₎Man₃GlcNAc₂, for example, GlcNAc₂ an₃GlcNAc₂, NANA₍₁.₄)GlcNAC₍i- ₄)Man₃GlcNAc2, and NA A₍i_-4)Gal ₍i-4)Man₃Glc AC₂; hybrid N-glycans are, in an embodiment of the invention, selected from the group consisting of Man₅GlcNAc₂ , GlcNAcMan₅GlcNAc₂ , GalGlcNAcMan₅GlcNAc₂, and

NANAGalGlcNAcMan₅GlcNAc₂; and high mannose N-glycans are, in an embodiment of the invention, selected from the group consisting of Man₆GlcNAc₂, Man₇GlcNAc₂ , Mang₈lcNAc₂, and Man₉GlcNAc₂ · The scope of the present invention includes such an isolated fungal host cell [e.g., Pichia) lacking full dihydroxyacetone synthase activity {e.g., due to knock out of DAS1 or DAS2) and including a

polynucleotide encoding a heterologous polypeptide (e.g., an immunoglobulin chain) .

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 are expressed, as discussed herein, and 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.

Sequences

The present invention includes fungal host cells in which DAS1 or DAS2 has been knocked-out and methods of use thereof as discussed herein. The identities of DASl and DAS2 are known in the art. In an embodiment of the present invention, a P. Pastoris

DASl/DAS2 gene locus comprises the nucleotide sequence:

CGTTATGTTGGATGAATGACTAGTATGCTGTTTGGGGAAATCAGGGTTGGGGCAGTCCTTTGAGTGG TACCAAATGACCAATGGTTGAGGACGGACGAATGTACAGTCCCGGGTCTACGAAATATGCATAAGAC TGTCTAACAACAAGAGGGGTTGGGGCGATCGCGCTAAACAACATCTGCTTTGTTGTACATTTTGTAC GTCATTCCAACTACCAACCTTCTCGGAAGCCAACCTCTTATCGGATAGAGCGGAACGGGAAGAACCC ATGGACGGAGGATAACAGACATTGGCCAGCGAGCGAGATTGAAGGAGGAGAAAAATCGTAAACTTGA TACACAATAACAGATTTGGAGGGGTGGTTTTTATCTAATTAATCTGCTCGCTCTTCCTCCTCTCCTC TCCTCACTCCACTTCTACGTTCATTCCCTTTCTGTACCCCCGTTCTGTGATGTAAGACGTGACGATG ATTGGTCAAATCTACAGTTCTGCTTATTCCCCCACCACCGCACCCTCATTGGGAGTGTTCAAGTGGT GCTGGCTGGTGTATCTCTCGATGTCGTATGATTCTGATAGGAGAATGTATGAGTTCTACGTTTGGCA TTGATGCCAGCTACTGCGCGCAATCCGCACCCAGCACCCCTTAATTCTTTTGATGATGCCCCGCTTC ACTGCTCGGGTGTGCTCTCGGGAGTATTTCCGATTAGCTCAACTCTCACGTTCAAAGTTAAACTTTA TTGACGAAGAGGAACTTTCTAAACGGCTGGACAGAAACTTAGATTAGGGAATGACGGTGTTTACACC TCCAGATTCTGTTCCAACACGATATGGACTTGTAGCTCGCTTCGATCAAATTTTTCAAGTGTGAACG TCTCCTGCAGCAGTCTTTCATCATACATATGAAAAATTTTTAAGGAGTTCTTTACCCCAGTTTTCAG ATTTTTTTTTCTCTCTTCAGCTGTACGCAGTACTAGAATCAGCGACCTATAAACACGAAACTACTAT ACAATCCAACACTAGCTGTGTATCTATTCCACTCTCTCCAATACTGTTTCCCCAATCCTACCACTAA CGTTCGGTTGAGCCCTCTCTATTACCCCATATTTTACTATATTTATCTTGTTGTCTTTTTATGCACG TATTATGCACATAGTATGTAGGTCGCAGAGGATTCACCCACATTGAAAAATGTTTCCTATAAATCAA ACTCCTCATATCTGCAGTGACAGGTTTCTCCCCCACAATCTTCGTCCCTTCAATATCCGGGCGTTGC CATTTCTGAGTGCAGCAGTGTTTCCTGCAAAGCCATCAAGAGGAGCTACATGCTCTCATTTTTTGGT TTTGTATGTCCGACGGGGTTCGTAAACTGGTTCCTCCTTTCCTTCCTGTTCCATTTATTGGTCAAAA AACAGGTCTTTCCTAAACCTTATGTCAATGACCTACCACATAGTGTTGCATCTGAAGATTGAGGGGG TGTACATCACCATCCCACCCTAGGATGTCCTACAGGTAAACAGATAGCCACTACGATGATCATCCAT AAACAAAAATCAAATTTAATAGACTTACACAACTACTAACCCGTTAGTGGCCAAATCTACTTATAGT TTGTCGTGCTTTGGTTTTCCCTTCAAATCGGTGAATTCGTAAAGGATTTGAGGACTAGCCTTAATAG AGTTGACGTATGCAGCAACCTTCTCACCAATCTTCTCAGGAGTGTATCCGAAGTATTTGTAGACATC CTCAACAGGAAGAGACTTACCAAAGGTGTTCATGGTGTAACCAGCAGTGGCGTATCTCTCCCATCCG TATGCGACATAGGCCTCAACAACGACAGTTGGAACCTCTCCTCTTCTAAGGACAGAACGTCTGTATG CCAGGGATTGTTGGTCAAACAGTCTCTGACATGGGAATGACAGAACTCTGACCTTCCATCCCTTCTG TTGTCTTAGCAAACGAGCAGTTTTGACGGCAAACTCCAATTCGGAACCAGCACCAATTAATTGGACG TCTGGCTTACCCTCACAGTCTTCAACGACGTAACCACCTCTTTTGGCTCCCTCAGCCGAAGTCTTAC CTGGGTATTGCTCAACCTCGTGTCTGGACAGAGAGAACAAGGTGGAGTGCTCGAGCTCAACAGCTAC TTCAAACAGAGCTGCAACCTCGGTAGCATCGGCTGGTCTAATGTAGTAGAAGTTGGGCATAGCTCTG AATAATGAAGACAAAGCAATAGGCTGGTGCGTTGGACCATCTTCACCAGCTCCGATGGAGTCGTGTG TAGCAATGTGAATTGCTTTCAACTCTTGAAGTGCAGCCATACGCAAGGCAGGTGCTGCATACAGGTA GAACATGTAGAAAGTTGAGGTAATAGGCAAGAAAGTACCCTTGTTGTATGCAGCCAAACCATTGGCA ATAGCACACATAGAGTGTTCTCTGATACCAAACTCAATATATCTACCAGAGTAGTCACCACCCAATC CACAGAAAGTTTGTAACTTAGGGTTGAAGAAGTACTTAACTCCTCCCCAGTTCAAAAGAATGGACAC GGACAAGTCACCAGAACCAGCAATAACCTGAGGAAGGTTTTGTCCCAGAGATCTAACAATTTCTCTA GCAGAGGTTCTGGTAGCAGTTGGCTCGGTTGGTTTGTCCTGTGGAATGAAACTCTTCCAGTTCTTTG GAAGTTCACCTCTAATACGGGCCTTTAATTCCTCACCTTCTTGAGGATAGTTCTTAACGTACTCATC CAAAAGTTTCTTCCAGTTAGCAACTAGTTGATCACCCTCGGCTGGTTTTTCAGCAAAGAAATCATAG ACCTCCTGTGGGAACCAGAACTTTCTAGCGACATCGAAACCGTACTTGGCCTTCAACTCCCGGATAC CTTCCTCACCAAGAGCAGAACCGTGAGCAGCGTGGTGGTTACCGAAAGCAGAATCCTGTCCAATTTC AGTTCTAACGTTGATCAGAGTTGGTCTCTCATTCTCAGCCTTGGCCCATTCGATGGCCTTGACAAGG GTAGCAACATCTCTAGAACCATTCTCGACTTCAATGACATTCCAGTTCTGAGCTCTAAACTTAGCAG AAATGTCTTCGGTGTTGTTAACATCGACGGAACCATCACAACAAACCTGGTTGTTGTCGTAGATCAC AATAAGGTTGTCCAAGGCCAAGTGACCAGCTAAGGAAATCGATTCCAAAGCAGGTCCCTCTTGCAAA CAAGCATCACCAACAATAGCATAGATAGTGTTGTCAACGACAGGGAAGCCAGGTCTGTTGTAAGTAG CGGCCAGGTTCTTTGAACCAATGGCCATACCGACAGCGTTAGAGATACCTTGTCCCAGGGGACCAGT GGTAACCTCAACAGCAGGGTTCTCAATTTCAGGGTGTCCAGGAGTCAATGAGTGATAATCGGAAGAG TGGTAAGATTGAAGTTGCTTGACAGTCATCTCCTTCAAACCAGTTAAGTGCTGGAACAAGTATTGGA ACAGACAGACGTGACCGTTTGACAAGACAAAACGATCTCTGTTGAAGTAGTCTGGATCATTTGGAGC GTACTTCATCTGGTACTTCCACAGAGCGATACCAATGGCGACCATACCCATGGCAGAACCAGGGTGA CCACCACCATACTGTTCGACTAAGTCGAGAACGTAACAACGGAAGGTTTTGATGACCAATTCATGAA TGTCATCTTGTGTCGATACTGCTTTTGGAATTCTAGCCATTTTTGATGTTTGATAGTTTGATAAGAG TGAACTTTAGTGTTTAGAGGGGTTATAATTTGTTGTAACTGGTTTTGGTCTTAAGTTAAAACGAACT TGTTATATTAAACACAACGGTCACTCAGGATACAAGAATAGGAAAGAAAAACTTTAAACTGGGGACA TGTTGTCTTTATATAATTTGGCGGTTAACCCTTAATGCCCGTTTCCGTCTCTTCATGATAACAAAGC TGCCCATCTATGACTGAATGTGGAGAAGTATCGGAACAACCCTTCACTAAGGATATCTAGGCTAAAC TCATTCGCGCCTTAGATTTCTCCAAGGTATCGGTTAAGTTTCCTCTTTCGTACTGGCTAACGATGGT GTTGCTCAACAAAGGGATGGAACGGCAGCTAAAGGGAGTGCATGGAATGACTTTAATTGGCTGAGAA AGTGTTCTATTTGTCCGAATTTCTTTTTTCTATTATCTGTTCGTTTGGGCGGATCTCTCCAGTGGGG GGTAAATGGAAGATTTCTGTTCATGGGGTAAGGAAGCTGAAATCCTTCGTTTCTTATAGGGGCAAGT ATACTAAATCTCGGAACATTGAATGGGGTTTACTTTCATTGGCTACAGAAATTATTAAGTTTGTTAT GGGGTGAAGTTACCAGTAATTTTCATTTTTTCACTTCAACTTTTGGGGTATTTCTGTGGGGTAGCAT AGCTTGACAGGTAATATGATGTACTATGGGATAGGCAAGTCTTGTGTTTCAGATACCGCCAAACGTT AAATAGGACCCTCTTGGTGACTTGCTAACTTAGAAAGTCATGCCCAGGTGTTACGTAATCTTACTTG GTATGACTTTTTGAGTAACGGACTTGCTAGAGTCCTTACCAGACTTCCAGTTTAGCAAACCACAGAT TGATCTGTCCTCTGGCATATCTCAAACCAATCAACACCCGTAACCCTTTCATGAAACAACTCTAGAA TGCGTCTTATCAACAGGATTGCCCAAAACAGTAATTGGGGCGGTGGAATCTACATGGGAGTTCCATC GTTGTCTCGGTTTTTCTCCCTATAAGCTACTCTGGAGACGAAGTAACTAACACCCTCAAATATCATT ATGTCCTGGTCAGGGTTCAAGAAAGCCGTCAATAGAGCTGGAACGCAGGTCCTTATGAAGACAAACC ATCTTGATGAGAGTCTGGATGAAGAGTTTGATTTCCAGGAGAAGAACTTCCGGATTATCCAACAATT TACTCAAGAGCTCTACAATCGACTTTCAAGCTTATTGGAAAATCATCATAGTTGTCTAAAGGCTAAT CTAGCCGTTGCTACCACTTTGAACTCATATTATGGAACCTCCACTACGGATGGATTTGAAGGAAAAT ATCTGGAGATCGTCAACAGGATAAAAGACGATGTGTTACCCAATTCAGTGGAACCGTTCAATTATAC AATATTGCAACCGTTAGAGACTCTTAAACAGTACAATGAAGAGTTTGACTTGTTAATAAAAAAACGT TATAGAAAGAAATTGGACTACGATATGCTCCAATCCAAATTGTCAAAATTGACCACCGAAAAAGAAC AATTGGAATTTGACAAGAGGAACAACTCACTAGATTCTCAAACGGAGCGTCACCTAGAGTCAGTTTC CAAGTCAATTACAGAAAGTTTGGAAACAGAAGAGGAGTATCTACAATTGAATTCCAAACTTAAAGTC GAGCTGTCCGAATTCATGTCGCTAAGGCTTTCTTACTTGGACCCCATTTTTGAAAGTTTCATTAAAG TTCAGTCAAAAATTTTCATGGACATTTATGACACATTAAAGAGCGGACTACCTTATGTTGATTCTCT ATCCAAAGAGGATTATCAGTCCAAGATCTTGGACTCTAGAATAGATAACATTCTGTCGAAAATGGAA GCGCTGAACCTTCAAGCTTACATTGATGATTAGAGCAATGATATAAACAACAATTGAGTGACAGGTC TACTTTGTTCTCAAAAGGCCATAACCATCTGTTTGCATCTCTTATCACCACACCATCCTCCTCATCT GGCCTTCAATTGTGGGGAACAACTAGCATCCCAACACCAGACTAACTCCACCCAGATGAAACCAGTT GTCGCTTACCAGTCAATGAATGTTGAGCTAACGTTCCTTGAAACTCGAATGATCCCAGCCTTGCTGC GTATCATCCCTCCGCTATTCCGCCGCTTGCTCCAACCATGTTTCCGCCTTTTTCGAACAAGTTCAAA TACCTATCTTTGGCAGGACTTTTCCTCCTGCCTTTTTTAGCCTCAGGTCTCGGTTAGCCTCTAGGCA AATTCTGGTCTTCATACCTATATCAACTTTTCATCAGATAGCCTTTGGGTTCAAAAAAGAACTAAAG CAGGATGCCTGATATATAAATCCCAGATGATCTGCTTTTGAAACTATTTTCAGTATCTTGATTCGTT TACTTACAAACAACTATTGTTGATTTTATCTGGAGAATAATCGAACAAAATGGCTAGAATTCCCAAA GCAGTTTCTTACAATGATGACATCCATGACTTGGTCATCAAAACCTTCCGTTGTTACGTTCTCGACT TAGTCGAACAGTATGGTGGTGGTCACCCTGGTTCTGCCATGGGTATGGTCGCCATTGGTATCGCTCT GTGGAAGTACCAGATGAAGTACGCTCCAAATGATCCAGACTACTTCAACAGAGATCGTTTTGTCTTG TCAAACGGTCACGTCTGTTTGTTCCAATACTTGTTCCAGCACTTAACTGGTTTGAAGGAGATGACTG TCAAGCAACTTCAATCTTACCACTCTTCCGATTATCACTCATTGACTCCTGGACACCCTGAAATTGA GAACCCTGCTGTTGAGGTTACCACTGGTCCCCTGGGACAAGGTATCTCTAACGCTGTCGGTATGGCC ATTGGTTCAAAGAACCTGGCCGCTACTTACAACAGACCTGGCTTCCCTGTCGTTGACAACACTATCT ATGCTATTGTTGGTGATGCTTGTTTGCAAGAGGGACCTGCTTTGGAATCGATTTCCTTAGCCGGTCA CTTGGCCTTGGACAACCTTATTGTGATCTACGACAACAACCAGGTTTGTTGTGATGGTTCCGTCGAT GTTAACAACACCGAAGACATCTCCGCAAAGTTCAGAGCTCAGAACTGGAATGTTATCGACATTGTAG ACGGTTCTAGAGATGTCGCTACCATTGTCAAGGCTATCGATTGGGCCAAGGCTGAGACTGAGAGACC AACTCTGATCAA.CGTTAGAACTGAAATTGGACAGGATTCTGCTTTCGGTAACCACCACGCTGCTCAC GGTTCTGCTCTAGGTGAGGAAGGTATCCGGGAGTTGAAGACTAAGTACGGTTTTAACCCTGCCCAAA AGTTCTGGTTCCCTAAAGAAGTATACGACTTCTTTGCTGAGAAACCAGCTAAAGGTGACGAGTTAGT AAAGAACTGGAAAAAGTTAGTTGATAGCTATGTCAAAGAGTACCCTCGTGAGGGACAAGAGTTCCTT TCTCGTGTTAGAGGTGAGCTTCCAAAGAACTGGAGAACTTACATTCCTCAAGACAAGCCTACCGAAC CAACCGCCACCAGAACCTCTGCTAGAGAAATTGTTAGGGCCCTTGGAAAGAACCTTCCTCAAGTTAT TGCCGGTTCCGGTGACTTATCTGTCTCAATTCTTTTGAACTGGGACGGAGTGAAGTACTTCTTCAAC CCTAAGTTACAGACTTTCTGTGGATTAGGTGGTGACTACTCTGGTAGATATATTGAGTTTGGTATCA GAGAACACTCTATGTGTGCTATTGCCAACGGTTTGGCTGCATACAACAAGGGTACTTTCTTGCCTAT TACCTCTACCTTCTACATGTTCTACCTGTATGCAGCACCTGCCTTGCGTATGGCTGCTCTTCAAGAG TTGAAAGCGATTCACATTGCTACACACGACTCTATTGGAGCTGGTGAAGATGGTCCAACCCACCAGC CTATTGCTTTGTCTTCATTATTCAGAGCTATGCCCAACTTCTACTACATGAGACCAGCCGATGCTAC CGAAGTTGCAGCTCTGTTTGAAGTGGCTGTTGAGCTTGAACACTCCACATTGCTTTCTCTGTCCAGA CACGAGGTTGACCAATACCCAGGTAAGACTTCTGCCCAAGGAGCCAAAAGAGGTGGTTACGTTGTTG AAGACTGCGAAGGAAAGCCAGATGTGCAACTGATCGGAACTGGTTCCGAGTTGGAATTCGCTATTAA GACTGCTCGTTTGCTAAGACAACAGAAGGGATGGAAGGTCAGAGTTCTGTCATTCCCATGTCAGAGA TTGTTTGACGAGCAGTCTATTACTTACAGACGTTCCGTCCTTAGAAGAGGAGAAGTTCCAACTGTCG TTGTTGAGGCCTATGTCGCATACGGATGGGAGAGATACGCCACTGCTGGTTACACCATGAACACCTT CGGTAAGTCTCTTCCTGTTGAGGATGTCTACAAATACTTCGGATACACTCCTGAGAAGATTGGTGAG AGAGTGGTTCAATATGTCAACTCTATCAAGGCTAGTCCTCAAATCCTTTACGAATTCCACGACTTGA AGGGAAAACCAAAGCATGACAAGTTGTAAACGGGAAGTCTTTACAGTTTTAGTTAGGAGCCCTTATA TATGACAGTAATGCTAGTACGTTTTGTTTTGTTTAATTAATAACTTAGTTTATGTTAGCCTAGTATA GACTCCATCAATTTTTTTTGTTATTACGTAAGCCGCGATGATAATATCTGATGAAAAATTCCTATCA GAAAATAATTTATCAAAAGTTTCATGCGATATGAGACTAAGTAGAATAGGGACTCCCAAAGTGTCAG TCACAAGGGTCATTCCCGTTCGTAATGTGGTGATAGCGAGGAGAAAACCTGTCAGAGCAAGTAACAC CGACGCAAAGACATGGCTAATGAAAGAAGAGCAGAGAAGAATAAGACAGAAGGAGCAGGAGATGAAA CAAAGGCTAGAGGAACTAGAAAGGTTCAAAACAAAAGTACAGAAATCATATATAAGGAAAGAGGATA GGCATTTGGCACAAGAGATAGAAAAGGATCTTGACATAATCACTGATGATTACAATTTGGACAGCGA TGTAGACCTGGTCTTTGGAGAACTCATGCAAGCAGAAGAGCAGCCCAAATCGTTGAAGAGTTTACCA GGAGCCAGTGATGCAAGTGATAACACAGATAAATCGGTGGAACTGTTTTCCTTTCCATCTCCGAATC TGACGCTACCTGAAAAGGTGATACACCATATTGGGCCACTGGTGAAGCACATCAGTAATCCTGAAAA CATTCAATGGGGGAGACTTTTACTGGATTTGGAAAAAAATCAGGGGTTTAACGGTCTATCTGCCGTA GATGTTACCAGACTGATTCAAAATATACCCAAAGAGGAAAAATATCAGCATATGTCTTTGATTCATG AAATGATGTTTAACAGTGGGATCAGTCCTGATCGGTACTTGACAGATTTGATGATGACTGCCTTCTC TGAAAGGAGCTACTATGAACCATTGGTTGAGGCTCTTTTCCAAGACTATGACATCAATGGCTGGGCT CCAACAGATTATACTTTTGGA

(SEQ ID NO: 1)

In an embodiment of the invention, the P.pastoris DAS1 gene (Pp03g03500 ; PAS_chr3_0834 ) comprises the nucleotide sequence:

ATGGCTAGAATTCCAAAAGCAGTATCGACACAAGATGACATTCATGAATTGGTCATCAAAACCTTCC GTTGTTACGTTCTCGACTTAGTCGAACAGTATGGTGGTGGTCACCCTGGTTCTGCCATGGGTATGGT CGCCATTGGTATCGCTCTGTGGAAGTACCAGATGAAGTACGCTCCAAATGATCCAGACTACTTCAAC AGAGATCGTTTTGTCTTGTCAAACGGTCACGTCTGTCTGTTCCAATACTTGTTCCAGCACTTAACTG GTTTGAAGGAGATGACTGTCAAGCAACTTCAATCTTACCACTCTTCCGATTATCACTCATTGACTCC TGGACACCCTGAAATTGAGAACCCTGCTGTTGAGGTTACCACTGGTCCCCTGGGACAAGGTATCTCT AACGCTGTCGGTATGGCCATTGGTTCAAAGAACCTGGCCGCTACTTACAACAGACCTGGCTTCCCTG TCGTTGACAACACTATCTATGCTATTGTTGGTGATGCTTGTTTGCAAGAGGGACCTGCTTTGGAATC GATTTCCTTAGCTGGTCACTTGGCCTTGGACAACCTTATTGTGATCTACGACAACAACCAGGTTTGT TGTGATGGTTCCGTCGATGTTAACAACACCGAAGACATTTCTGCTAAGTTTAGAGCTCAGAACTGGA ATGTCATTGAAGTCGAGAATGGTTCTAGAGATGTTGCTACCCTTGTCAAGGCCATCGAATGGGCCAA GGCTGAGAATGAGAGACCAACTCTGATCAACGTTAGAACTGAAATTGGACAGGATTCTGCTTTCGGT AACCACCACGCTGCTCACGGTTCTGCTCTTGGTGAGGAAGGTATCCGGGAGTTGAAGGCCAAGTACG GTTTCGATGTCGCTAGAAAGTTCTGGTTCCCACAGGAGGTCTATGATTTCTTTGCTGAAAAACCAGC CGAGGGTGATCAACTAGTTGCTAACTGGAAGAAACTTTTGGATGAGTACGTTAAGAACTATCCTCAA GAAGGTGAGGAATTAAAGGCCCGTATTAGAGGTGAACTTCCAAAGAACTGGAAGAGTTTCATTCCAC AGGACAAACCAACCGAGCCAACTGCTACCAGAACCTCTGCTAGAGAAATTGTTAGATCTCTGGGACA AAACCTTCCTCAGGTTATTGCTGGTTCTGGTGACTTGTCCGTGTCCATTCTTTTGAACTGGGGAGGA GTTAAGTACTTCTTCAACCCTAAGTTACAAACTTTCTGTGGATTGGGTGGTGACTACTCTGGTAGAT ATATTGAGTTTGGTATCAGAGAACACTCTATGTGTGCTATTGCCAATGGTTTGGCTGCATACAACAA GGGTACTTTCTTGCCTATTACCTCAACTTTCTACATGTTCTACCTGTATGCAGCACCTGCCTTGCGT ATGGCTGCACTTCAAGAGTTGAAAGCAATTCACATTGCTACACACGACTCCATCGGAGCTGGTGAAG ATGGTCCAACGCACCAGCCTATTGCTTTGTCTTCATTATTCAGAGCTATGCCCAACTTCTACTACAT TAGACCAGCCGATGCTACCGAGGTTGCAGCTCTGTTTGAAGTAGCTGTTGAGCTCGAGCACTCCACC TTGTTCTCTCTGTCCAGACACGAGGTTGAGCAATACCCAGGTAAGACTTCGGCTGAGGGAGCCAAAA GAGGTGGTTACGTCGTTGAAGACTGTGAGGGTAAGCCAGACGTCCAATTAATTGGTGCTGGTTCCGA ATTGGAGTTTGCCGTCAAAACTGCTCGTTTGCTAAGACAACAGAAGGGATGGAAGGTCAGAGTTCTG TCATTCCCATGTCAGAGACTGTTTGACCAACAATCCCTGGCATACAGACGTTCTGTCCTTAGAAGAG GAGAGGTTCCAACTGTCGTTGTTGAGGCCTATGTCGCATACGGATGGGAGAGATACGCCACTGCTGG TTACACCATGAACACCTTTGGTAAGTCTCTTCCTGTTGAGGATGTCTACAAATACTTCGGATACACT CCTGAGAAGATTGGTGAGAAGGTTGCTGCATACGTCAACTCTATTAAGGCTAGTCCTCAAATCCTTT ACGAATTCACCGATTTGAAGGGAAAACCAAAGCACGACAAACTATAA

(SEQ ID NO: 2)

In an embodiment of the invention, the P. Pastoris DAS2 gene

(Pp03g03520 ; PAS_chr3_0832 ) comprises the nucleotide sequence:

ATGGCTAGAATTCCCAAAGCAGTTTCTTACAATGATGACATCCATGACTTGGTCATCAAAACCTTCC GTTGTTACGTTCTCGACTTAGTCGAACAGTATGGTGGTGGTCACCCTGGTTCTGCCATGGGTATGGT CGCCATTGGTATCGCTCTGTGGAAGTACCAGATGAAGTACGCTCCAAATGATCCAGACTACTTCAAC AGAGATCGTTTTGTCTTGTCAAACGGTCACGTCTGTTTGTTCCAATACTTGTTCCAGCACTTAACTG GTTTGAAGGAGATGACTGTCAAGCAACTTCAATCTTACCACTCTTCCGATTATCACTCATTGACTCC TGGACACCCTGAAATTGAGAACCCTGCTGTTGAGGTTACCACTGGTCCCCTGGGACAAGGTATCTCT AACGCTGTCGGTATGGCCATTGGTTCAAAGAACCTGGCCGCTACTTACAACAGACCTGGCTTCCCTG TCGTTGACAACACTATCTATGCTATTGTTGGTGATGCTTGTTTGCAAGAGGGACCTGCTTTGGAATC GATTTCCTTAGCCGGTCACTTGGCCTTGGACAACCTTATTGTGATCTACGACAACAACCAGGTTTGT TGTGATGGTTCCGTCGATGTTAACAACACCGAAGACATCTCCGCAAAGTTCAGAGCTCAGAACTGGA ATGTTATCGACATTGTAGACGGTTCTAGAGATGTCGCTACCATTGTCAAGGCTATCGATTGGGCCAA GGCTGAGACTGAGAGACCAACTCTGATCAACGTTAGAACTGAAATTGGACAGGATTCTGCTTTCGGT AACCACCACGCTGCTCACGGTTCTGCTCTAGGTGAGGAAGGTATCCGGGAGTTGAAGACTAAGTACG GTTTTAACCCTGCCCAAAAGTTCTGGTTCCCTAAAGAAGTATACGACTTCTTTGCTGAGAAACCAGC TAAAGGTGACGAGTTAGTAAAGAACTGGAAAAAGTTAGTTGATAGCTATGTCAAAGAGTACCCTCGT GAGGGACAAGAGTTCCTTTCTCGTGTTAGAGGTGAGCTTCCAAAGAACTGGAGAACTTACATTCCTC AAGACAAGCCTACCGAACCAACCGCCACCAGAACCTCTGCTAGAGAAATTGTTAGGGCCCTTGGAAA GAACCTTCCTCAAGTTATTGCCGGTTCCGGTGACTTATCTGTCTCAATTCTTTTGAACTGGGACGGA GTGAAGTACTTCTTCAACCCTAAGTTACAGACTTTCTGTGGATTAGGTGGTGACTACTCTGGTAGAT ATATTGAGTTTGGTATCAGAGAACACTCTATGTGTGCTATTGCCAACGGTTTGGCTGCATACAACAA GGGTACTTTCTTGCCTATTACCTCTACCTTCTACATGTTCTACCTGTATGCAGCACCTGCCTTGCGT ATGGCTGCTCTTCAAGAGTTGAAAGCGATTCACATTGCTACACACGACTCTATTGGAGCTGGTGAAG ATGGTCCAACCCACCAGCCTATTGCTTTGTCTTCATTATTCAGAGCTATGCCCAACTTCTACTACAT GAGACCAGCCGATGCTACCGAAGTTGCAGCTCTGTTTGAAGTGGCTGTTGAGCTTGAACACTCCACA TTGCTTTCTCTGTCCAGACACGAGGTTGACCAATACCCAGGTAAGACTTCTGCCCAAGGAGCCAAAA GAGGTGGTTACGTTGTTGAAGACTGCGAAGGAAAGCCAGATGTGCAACTGATCGGAACTGGTTCCGA GTTGGAATTCGCTATTAAGACTGCTCGTTTGCTAAGACAACAGAAGGGATGGAAGGTCAGAGTTCTG TCATTCCCATGTCAGAGATTGTTTGACGAGCAGTCTATTACTTACAGACGTTCCGTCCTTAGAAGAG GAGAAGTTCCAACTGTCGTTGTTGAGGCCTATGTCGCATACGGATGGGAGAGATACGCCACTGCTGG TTACACCATGAACACCTTCGGTAAGTCTCTTCCTGTTGAGGATGTCTACAAATACTTCGGATACACT CCTGAGAAGATTGGTGAGAGAGTGGTTCAATATGTCAACTCTATCAAGGCTAGTCCTCAAATCCTTT ACGAATTCCACGACTTGAAGGGAAAACCAAAGCATGACAAGTTGTAA

(SEQ ID NO: 3)

In an embodiment of the invention, the P. pastoris Daslp protein comprises the amino acid sequence:

ARIPKAVSTQDDIHELVIKTFRCYVLDLVEQYGGGHPGSAMG VAIGIALWKYQMKYAPNDPDYFN RDRFVLSNGHVCLFQYLFQHLTGLKEMTVKQLQSYHSSDYHSLTPGHPEIENPAVEVTTGPLGQGIS NAVGMAIGSKNLAATYNRPGFPWDNTIYAIVGDACLQEGPALESISLAGHLALDNLIVIYDNNQVC CDGSVDVNNTEDISAKFRAQNWNVIEVENGSRDVATLVKAIEWAKAENERPTLIN^

NHHAAHGSALGEEGIRELKAKYGFDVARKFWFPQEWDFFAEKPAEGDQLVANWKKLLDEYVKNYPQ EGEELKARIRGELPKN KSFIPQDKPTEPTATRTSAREIVRSLGQNLPQVIAGSGDLSVSILLNWGG VKYFFNPKLQTFCGLGGDYSGRYIEFGIREHSMCAIANGLAAYNKGTFLPITSTFYMFYLYAAPALR MAALQELKAIHIATHDSIGAGEDGPTHQPIALSSLFRA PNFYYIRPADATEVAALFEVAVELEHST LFSLSRHEVEQYPGKTSAEGAKRGGYWEDCEGKPDVQLIGAGSELEFAVKTARLLRQQKGWKVRVL SFPCQRLFDQQSLAYRRSVLRRGEVPTWVEAYVAYGWERYATAGYTMNTFGKSLPVEDVYKYFGYT PEKIGEKVAAYVNSIKASPQILYEFTDLKGKPKHDKL

(SEQ ID NO: 4)

In an embodiment of the invention, the P. pastoris Das2p protein comprises the amino acid sequence:

MARIPKAVSYNDDIHDLVIKTFRCYVLDLVEQYGGGHPGSAMGMVAIGIALWKYQ KYAPNDPDYFN RDRFVLSNGHVCLFQYLFQHLTGL EMTVKQLQSYHSSDYHSLTPGHPEIENPAVEVTTGPLGQGIS NAVG AIGSKNLAATYNRPGFPVVDNTIYAIVGDACLQEGPALESISLAGHLALDNLIVIYDNNQVC CDGSVDVNNTEDISAKFRAQNW3WIDIVDGSRDVATIVAIDWAKAETERPTLINVRTEIGQDSAFG NHHAAHGSALGEEGIRELKTKYGFNPAQKF FPKEVYDFFAEKPAKGDELVKNWKKLVDSYVKEYPR EGQEFLSRVRGELPKN RTYIPQDKPTEPTATRTSAREIVRALGKNLPQVIAGSGDLSVSILLNWDG V YFFNPKLQTFCGLGGDYSGRYIEFGIREHS CAIANGLAAYNKGTFLPITSTFYMFYLYAAPALR MAALQELKAIHIATHDSIGAGEDGPTHQPIALSSLFRAMPNFYY RPADATEVAALFEVAVELEHST LLSLSRHEVDQYPGKTSAQGAKRGGYWEDCEGKPDVQLIGTGSELEFAIKTARLLRQQKG KVRVL SFPCQRLFDEQSITYRRSVLRRGEVPTWVEAYVAYGWERYATAGYTMNTFGKSLPVEDVYKYFGYT PEKIGERWQYVNSIKASPQILYEFHDLKGKPKHDKL

(SEQ ID NO: 5)

In an embodiment of the invention, the P. pastoris Fldlp protein (Pp03g01420 ; PAS_chr3_1028) comprises the amino acid sequence :

MSTEGQIIKCKAAVAWEAGKDLSIEEIEVLPPRAHEVRVKVEFTGVCHTDAYTLSGADAEGSFPWF GHEGAGWESVGEGVESVKVGDSWLLYTPECRECKFCLSGKTNLCGKIRATQGKGLLPDGTSRFRC KGKDLFHY GCSSFSQYTWADISWKVQDEAPKDKTCLLGCGVTTGYGAAINTAKISKGDKIGVFG AGCIGLSVIQGAVSKGASEIIVIDINDSKKAADQFGATKFVNPTTLPEGTNIVDYLIDITDGGFDY TFDCTGNVQVMRNALESCHKGWGESIIIGVAAAGKEISTRPFQLVTGRVWRGCAFGGIKGRTQMPSL VQDYLDGKIKVDEFI HRHDLDNI KAFHDMHAGNCIRAVIT H

(SEQ ID NO: 6) In an embodiment of the invention, the P. pastoris FLD1 gene

Pp03g01420; PAS_chr3_1028 comprises the nucleotide sequence:

ATGTCTACCGAAGGTCAAGTAAGTTCAATCAAAGTAATTGTTTGGGAGGG AAGAAGATTGTTTTATTGCGAACCTTTCAATATCTTACCCGACTAAATAA CCATTACAGTGAATTTTTTACTAACTATATAGATCATCAAATGTAAGGCA GCTGTTGCCTGGGAGGCAGGAAAGGATCTCTCTATTGAGGAGATTGAGGT TCTTCCTCCAAGAGCCCATGAAGTTAGAGTGAAAGTGGAATTCACTGGTG TATGCCACACTGATGCTTACACGCTTTCTGGTGCAGATGCAGAGGGAAGT TTCCCTGTTGTGTTCGGCCATGAAGGTGCTGGTGTTGTCGAGTCAGTTGG AGAAGGTGTTGAGTCCGTGAAGGTTGGGGATTCTGTAGTGCTTCTGTACA CTCCTGAGTGCAGAGAGTGCAAGTTCTGTCTGTCTGGTAAGACGAACCTC TGTGGTAAAATCAGAGCCACCCAGGGTAAAGGTTTGTTACCAGACGGGAC TTCTCGTTTCCGTTGTAAGGGCAAGGATTTGTTTCACTATATGGGATGTT CTTCCTTTTCTCAATACACTGTGGTGGCTGACATCTCAGTGGTTAAAGTC CAAGACGAAGCTCCTAAGGACAAGACATGTCTGTTGGGTTGTGGTGTTAC CACAGGGTACGGTGCTGCTATCAACACTGCTAAGATCTCTAAGGGTGACA AGATCGGTGTGTTTGGTGCTGGATGTATTGGATTATCTGTCATCCAAGGT GCAGTTTCCAAAGGTGCAAGCGAGATTATTGTAATTGACATCAATGATTC AAAGAAGGCATGGGCGGACCAATTTGGTGCAACTAAGTTTGTCAATCCTA CAACCTTACCAGAAGGTACCAATATTGTTGACTACTTGATTGATATCACT GACGGAGGCTTTGACTATACCTTCGACTGTACCGGTAATGTTCAAGTAAT GAGAAATGCACTTGAATCTTGCCACAAGGGTTGGGGTGAGTCGATCATCA TCGGTGTCGCTGCTGCTGGTAAAGAAATCTCTACCCGTCCTTTCCAGTTG GTTACTGGCAGAGTCTGGAGAGGATGCGCCTTTGGAGGTATCAAGGGACG TACTCAAATGCCATCTTTGGTTCAGGACTATCTTGATGGTAAGATTAAAG TTGACGAGTTTATCACACACAGACATGACCTGGACAACATCAACAAAGCA TTTCATGACATGCATGCTGGAAACTGTATTCGTGCTGTGATTACTATGCA CTAA

(SEQ ID NO: 7)

In an embodiment of the invention, the P. pastoris Fghlp protein (Pp03g03140 ; PAS_chr3_0867) comprises the amino acid sequence :

MSSITTSIFKV AEIQSFGGKLVKLQHKSDETKTDMDVNVYLPAQFFANGAKGKSLPVLL YLSGLTCTPNNASEKAFWQPYANKYGFAWFPDTSPRGLNIEGEHDSYDFGSGAGFYVDA TTEKWKDNYRMYSYWSELLPKLQADFPILNFDNISITGHSMGGYGALQLFLRNPGKFKS VSAFSPISNPTKAPWGEKCFSGYLGQDKSTWTQYDPTELIGKYQGPSDSSILIHVGKSDS FYFKDHQLLPENFLKASENSVFKGKVDLNLVDGYDHSYYFISSFTDVHAAHHAKYLGLN

(SEQ ID NO: 8)

In an embodiment of the invention, the P. pastoris FGH1 gene

(Pp03g03140 ; PAS_chr3_0867) comprises the amino acid sequence:

ATGTCATCAATTACTACTTCAATCTTCAAGGTAACAGCTGAAATCCAAAGTTTTGGGGGA AAGCTAGTCAAACTTCAACACAAGTCCGATGAGACGAAGACTGACATGGATGTGAACGTC TACCTTCCAGCTCAATTCTTTGCCAATGGAGCCAAGGGAAAATCATTACCAGTTCTACTT TATTTGAGTGGTCTGACTTGCACTCCCAACAATGCCTCAGAGAAGGCATTTTGGCAACCA TATGCAAATAAGTACGGTTTTGCTGTGGTTTTCCCGGATACTTCACCCAGAGGGCTCAAC ATCGAAGGAGAGCACGACTCTTATGATTTTGGATCCGGTGCCGGGTTCTACGTGGATGCC ACTACTGAGAAATGGAAGGATAATTATAGAATGTACAGTTATGTTAACTCGGAATTGCTA CCCAAATTGCAGGCTGACTTCCCAATTCTAAACTTTGACAATATTTCAATCACGGGCCAC TCCATGGGAGGTTACGGAGCTTTACAGTTATTCTTGAGAAACCCGGGAAAATTCAAGTCG GTTTCCGCATTTTCTCCAATCTCCAACCCCACTAAAGCCCCATGGGGTGAGAAGTGCTTC

TCTGGATACCTGGGACAGGACAAGTCCACTTGGACTCAGTACGACCCAACCGAATTGATT GGAAAATACCAAGGCCCCTCAGATTCCAGCATTTTGATTCACGTTGGAAAGAGTGATTCG TTCTACTTCAAGGACCACCAGCTGCTACCTGAGAACTTCTTGAAGGCTTCAGAGAACTCT GTGTTCAAGGGAAAAGTGGACTTGAACTTGGTAGATGGCTATGACCATTCTTACTACTTT ATCTCTTCATTCACAGACGTTCATGCTGCTCACCATGCAAAGTATTTGGGGTTAAACTAG

(SEQ ID NO: 9)

In an embodiment of the invention, the P. pastoris FMDHp protein (Pp03g02400; PAS_chr3_0932) comprises the amino acid sequence :

MKIVLVLYSAGKHAADEPKLYGCIENELGIRQWLEKGGHELVTTSDKEGENSELEKHIPD ADVIISTPFHPAYITKERIQKAKKLKLLWAGVGSDHIDLDYIEQNGLDISVLEVTGSNV VSVAEHWMTILNLVRNFVPAHEQIVNHG DVAAIAKDAYDIEGKTIATIGAGRIGYRVL ERLVAFNPKELLYYDYQGLPKEAEEKVGARRVDTVEELVAQADWTVNAPLHAGTKGLVN KELLSKFKKGAWLVNTARGAICNAQDVADAVASGQLRGYGGDVWFPQPAPKDHP RDMRN KYGYGNAMTPHYSGTTLDAQVRYAEGTKNILNSFLTKKFDYRPQDVILLNGKYKTKAYGN DKKVA

(SEQ ID NO: 10)

In an embodiment of the invention, the P. pastoris FMDH gene

(Pp03g02400 ; PAS_chr3_0932 ) comprises the nucleotide sequence:

ATGAAAATCGTTCTCGTTTTGTACTCCGCTGGTAAGCACGCCGCCGATGAACCAAAGTTG TATGGTTGTATCGAAAATGAATTGGGTATTAGACAATGGCTTGAGAAGGGCGGCCATGAA TTGGTTACTACATCAGACAAAGAGGGTGAAAACTCTGAGTTAGAAAAGCACATTCCTGAC GCTGATGTGATTATTTCCACTCCATTCCATCCAGCCTACATCACGAAGGAGAGAATCCAA AAAGCCAAGAAGCTGAAGTTGTTGGTCGTTGCTGGTGTCGGTTCCGACCACATTGACTTG GACTACATTGAACAAAATGGCCTAGATATTTCGGTCCTAGAGGTTACTGGTTCCAACGTT GTTTCAGTGGCTGAGCATGTCGTTATGACTATATTGAACTTGGTGAGAAACTTTGTTCCA GCTCACGAGCAAATTGTTAACCACGGCTGGGACGTTGCTGCCATCGCCAAGGACGCCTAC GATATCGAAGGTAAGACCATCGCAACAATTGGTGCTGGAAGAATTGGTTACAGAGTCTTA GAGAGACTTGTGGCTTTCAACCCTAAGGAATTGTTGTACTACGACTACCAAGGTCTTCCA AAAGAGGCCGAGGAAAAAGTTGGTGCCAGAAGAGTCGACACTGTCGAGGAGCTGGTTGCT CAAGCCGATGTTGTTACCGTCAATGCCCCACTGCACGCAGGTACTAAGGGTTTAGTTAAC AAGGAGCTTCTGTCCAAGTTCAAGAAGGGTGCTTGGTTGGTTAACACAGCCAGAGGTGCC ATCTGCAATGCTCAAGATGTCGCTGATGCCGTTGCATCTGGTCAATTGAGAGGTTACGGT GGTGACGTCTGGTTCCCTCAGCCAGCTCCAAAGGACCATCCATGGAGAGATATGAGAAAC AAGTACGGATACGGAAACGCCATGACTCCTCATTACTCAGGTACCACTTTGGACGCCCAG GTCAGATATGCCGAAGGTACCAAGAACATCTTGAACTCATTCCTTACCAAGAAGTTTGAC TACAGACCTCAAGATGTCATTCTTTTGAACGGTAAGTACAAGACCAAGGCTTATGGTAAT GACAAAAAGGTCGCATAA

(SEQ ID NO: 11)

Expression Methods

The present invention encompasses methods for making a polypeptide (e.g., an immunoglobulin heavy and/or light chain or an antibody or antigen-binding fragment thereof) comprising

introducing, into an isolated fungal host cell of the present invention (e.g. , Pichia, e.g., Pichia pastoris) , in which, for example, DAS1 or DAS2 is knocked-out, a heterologous polynucleotide e.g. , operably linked to a promoter {e.g., a methanol -inducible promoter) ; and, culturing the host cell (e.g., in a liquid culture medium, e.g., YPD medium (e.g., comprising 1% yeast extract, 2% peptone, 2% glucose)), optionally in the presence of methanol, under conditions whereby the heterologous polynucleotide encoding the heterologous polypeptide is expressed, thereby producing the polypeptide. Expression of the polynucleotide may be induced when the promoter is methanol-inducible and the host cells are grown in the presence of methanol.

An expression system, comprising the fungal host cells of the present invention (e.g., dasl or das2) comprising the promoter operably linked to the heterologous polynucleotide, e.g., in an ectopic vector or integrated into the genomic DNA of the host cell, forms part of the present invention. A composition comprising the fungal host cell which includes the promoter operably linked to the heterologous polynucleotide in liquid culture medium also forms part of the present invention.

In one embodiment of the invention, a method for expressing a heterologous polypeptide, e.g., as discussed herein, does not comprising starving the fungal host cells of a nutrient such as a carbon source such as glycerol or glucose. Other embodiments include methods wherein the cells are starved. For example, the present invention comprises methods for expressing a polypeptide in a fungal glycosylation mutant host cell, e.g., as discussed herein, wherein the host cell comprises a promoter (e.g. , methanol- inducible) operably linked to a heterologous polynucleotide encoding the polypeptide wherein the host cell is or is not starved and is cultured in the presence of methanol.

In an embodiment of the invention, the heterologous

polynucleotide that is operably linked to the promoter is in a vector that comprises a selectable marker. In an embodiment of the invention, the fungal host cells (e.g. , dasl or das2) , e.g., Pichia cells, are grown in a liquid culture medium and cells including the vector with the selectable marker are selected for growth; e.g. , wherein the selectable marker is a drug resistance gene, such as the zeocin resistance gene, and the cells are grown in the presence of the drug, such as zeocin.

In an embodiment of the invention, heterologous polypeptide expression using a methanol -inducible promoter includes three phases, the glycerol batch phase, the glycerol fed-batch phase and the methanol fed-batch phase. First, in the glycerol batch phase (GBP), fungal host cells (e.g., dasl or das2) are initially grown on glycerol in a batch mode. In the second phase, the glycerol fed-batch phase (GFP) , a limited glycerol feed is initiated following exhaustion of the glycerol in the previous phase, and cell mass is increased to a desired level prior to methanol- induction. Furthermore, the methanol -inducible promoters are de- repressed during this phase due to the absence of excess glycerol. The third phase is the methanol fed-batch phase (MFP) , in which methanol is fed at a limited feed rate or maintained at some level to induce the methanol-inducible promoters for protein expression. A limited glycerol feed can be simultaneously performed for promoting production when necessary.

Accordingly, the present invention encompasses methods for making a heterologous polypeptide (e.g., an immunoglobulin) comprising introducing, into an isolated fungal host cell, for example, dasl or das2 (e.g., Pichia, such as Pichia pastoris) a heterologous polynucleotide encoding said polypeptide that is operably linked to a methanol -inducible promoter of the present invention 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, prior to the batch phase, an initial seed culture is grown to a high density (e.g., OD₆₀₀ of about 2 or higher) and the fungal host 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 fungal 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.

In an embodiment of the invention, the fungal host cells {e.g. , Pichia cells such as Pichia pastoris) are grown under any 1, 2, 3, 4, 5 or 6 of the following conditions:

• in a culture medium at a pH of about 5; and/or at a

temperature of about 30°C; and/or

• in the presence of any 1 or more trace minerals/nutrients such as copper, iodine, manganese, molybdenum, boron, cobalt, zinc, iron, biotin and/or sulfur, e.g. , CuS0₄, Nal, MnS0₄, Na₂Mo0₄, H₃BO₃, CoCl₂, ZnCl₂, FeS0₄, biotin and/or H₂S0₄ and/or

• in the presence of an anti-foaming agent {e.g. , silicone) ; and/or

• at an oxygen concentration of about 20% saturation or higher; and/or

• in a glycerol batch phase at a glycerol concentration of about 40 grams/liter; and/or

• in the methanol fed-batch phase at a methanol concentration of about 2 grams methanol/liter to about 5 grams methanol/liter (e.g., 2, 2.5, 3, 3.5, 4, 4.5 or 5) .

The present invention provides methods for making polypeptides, such as immunoglobulin chains, antibodies or antigen-binding fragments thereof having modified glycosylation patterns, for example, by expressing a polypeptide in a fungal host cell that introduces a given glycosylation pattern and/or by growing the fungal host cell under conditions wherein the glycosylation is introduced. Some of such host cells are discussed herein. For example, the invention provides methods for making a heterologous protein that is a glycoprotein comprising an N-glycan structure that comprises a Man₅GlcNAc₂ glycoform; comprising introducing a polynucleotide encoding the polypeptide wherein the polynucleotide is operably linked to a promoter of the present invention into a host cell and culturing the host cell under conditions wherein the polypeptide is expressed with the Man₅GlcNAc₂ glycoform and/or lacking fucose.

Examples

The present invention is intended to exemplify the present invention and not to be a limitation thereof. The methods and compositions disclosed below fall within the scope of the present invention. Example 1 ; Expression profiling of P. pastoris strains reveals differential induction of Methanol Assimilation Pathway Genes

Briefly, the P. pastoris wild type strain RRL-yll430 and two glycoengineered strains, YGLY8316 and YGLY8323, were cultivated in quadruplicate in 0.5L bioreactors (Sixfors multifermentation system; ATR Biotech, Laurel, MD) using a standard glycerol-to- methanol fed-batch protocol as described in Barnard et al . , 2010 (J. Ind. Microbiol. Biotechnol . 37:961-971). Samples were taken from each bioreactor during the glycerol phase at 50mg/ml of wet cell weight (batch) , during the starvation period at the end of batch (EOB) , and after 4+/-1 hours (4h MeOH) and 24+/-1 hours (24h MeOH) of methanol induction. Harvested cells were centrifuged, flash frozen, and the frozen cell pellets were then used for R A extraction and microarray hybridization using a custom-designed Agilent P. pastoris 15k 3.0 array (8xl5K) based upon an internal P. pastoris genome sequence for strain NRRL Y-11430. Intensity data were uploaded to Rosetta Resolver (Rosetta Biosoftware, Kirkland, WA) . Standard Resolver pipelines for the Agilent Single Color Error Model were used for data summarization and calling (Weng et al., Bioinformatics 22, 2006, 1111-1121; Davidson, promoters,

23011-US-PSP) . These models determined whether a particular gene exhibited differential expression for the ratio comparison specified, although such differential expression calls were typically made via ANOVA and t-test statistical tests that were also performed. In addition to these statistical tests,

clustering, PCA, and other operations were also performed upon the data using standard Resolver software methods (Rosetta Resolver User Guide, 2002, Kirkland, WA) .

To analyze the expression patterns of genes in the methanol pathway, first, a predicted gene list was identified via homology search using BlastP of protein sequences predicted to function in the methanol dissimilation and assimilation (pentose phosphate) pathways (see Hartner, 2008 and Figure 1) . These sequences were BlastP searched against a predicted ORF translation database generated from an internal genome sequence of P. pastoris strain NRRL-yll430. The sequences were all confirmed to be identified in the P. pastoris public genome database (Genbank, NCBI, Bethesda, MD) , and these genes are listed in Table 1. Intensity data generated from the experiment comparing wild type and

glycoengineered strains were then ratioed to batch in a strain- specific manner. The intensity ratios for the methanol utilization assimilation pathway genes were clustered against RRL-yll430 and YGLY8323 samples at batch and 24h MeOH and plotted (Figure 2) .

From these data it can be seen that most of the genes encoding members of the assimilation pathway are upregulated. Duplicated pairs of genes encoding potential isozymes exist at several steps in the pathway, and at least two predicted members of the pathway (TALI and FBA1) are encoded by nearly identical genes where one of the two genes is induced by methanol, but the other is not.

However, unlike the case in the baker's yeast S. cerevisiae, P. pastoris appears to contain only a single classical transketolase gene, TKL1, encoding a key central member of the pentose phosphate shunt and in methylotrophic yeasts, the methanol assimilation pathway. This enzyme is responsible for the reversible transfer of a ketol from a ketose (xylulose 5 -phosphate, fructose 6 -phosphate or sedoheptulose 7-phosphate) to an aldose (ribose 5 -phosphate, erythrose 4 -phosphate or glyceraldehyde 3 -phosphate) . The lack of induction of this gene supports a model where upon methanol induction TKL1 may play a role as a bottleneck in maintaining flux through the pathway and keeping the levels of upstream metabolites such as F6P and F1,6BP high (Figure 1) to support central carbon pathways for generation of cellular carbon containing components. 5 However, when flux in this pathway becomes limiting due to higher energy demands rather than transketolase activity, the levels of X5P (Figure 1) decline, formaldehyde cannot be efficiently

converted into DHA and excess formaldehyde escapes to the cytosol where it is oxidized via the dissimilation pathway to C0₂ while 0 generating NADH reducing equivalents to support energy demands.

Table 1 : Enzymes of the methanol utilization pathway

This balance of metabolite pools maintenance and energy generation efficiently balances the ability of a microorganism to utilize methanol and available nitrogen sources to generate metabolites to multiply and energy to support those processes.

However, the energy demands of a naive cell growing on methanol can differ substantially from a genetically engineered cell producing significant amounts of a heterologous secreted protein, such as a monoclonal antibody (mAb) . The inability to efficiently shift resources to meet the demands of producing, folding and secreting large amounts of such a protein can cause energy levels to become limiting, particularly when the protein is difficult to fold, resulting in ER stress (For review, see: Boyce, 2006) . This can result in reduction or abrogation of protein synthesis or in some cases in cell death. In an industrial protein production process this can result in reduced product yield or loss of product due to release of intracellular proteases. In support of this assertion it has been shown specifically that significant transcriptome changes can occur in P. pastoris upon over-expression of a

heterologously secreted protein (Gasser, 2007) .

Therefore, these expression data, combined with the previous knowledge of the impact of ER stress during heterologous protein production, support a hypothesis whereby specific fine-tuning of the methanol utilization assimilation and dissimilation pathways could improve the energy balance of genetically engineered cells producing a heterologously secreted protein, e.g. a mAb (Figure 3) .

Example 2 : Deletion of one of the two genes encoding

Dihydroxyacetone Synthase in P. pastoris .

Dihydroxyacetone synthase, DHAS or DAS, is a specialized transketolase-like enzyme, present in methylotrophic organisms, responsible for conversion of formaldehyde and the 5-C member of the pentose phosphate pathway, X5P, into the two 3-C members of core metabolic pathways, G3P and DHA (Figure 1) . This reaction is followed by several rearrangements to yield a net conversion of 3 moles of formaldehyde to 1 mole of G3P for 3 turns of the

assimilation pathway cycle. In P. pastoris, there are two

predicted genes encoding DAS, DAS1 and DAS2, which are linked tightly on Chromosome 3 (Figure 4) . To reduce flux in the methanol assimilatory pathway, one of the two DAS genes was deleted by homologous integration of the P. pastoris URA5 gene as a selectable marker. This was expected to shunt more formaldehyde into the dissimilatory pathway and thereby shift the balance of energy reserves when heterologous protein synthesis is initiated.

Knock-out or deletion of P. pastoris DASl was performed by first polymerase chain reaction amplification of the 5' and 3' regions adjacent to the DASl gene. The DASl 5' region was

amplified using primers RCD969 (5¹ -GAGCTCGGCCAGCTTGGCCGTTGTTTCATG AAAGGGTTACGGG-3 ' ) (SEQ ID NO: 12) and RCD970 (5'-

GTTTAAACTTTTGATGTTTGATAGTTTGATAA GAGTG-3 ' ) (SEQ ID NO: 13). This region was cloned into the Topo TA cloning vector (Invitrogen, Carlsbad, CA) and sequenced. The DASl 5' fragment was subcloned into plasmid pGLY24 using the Sacl/Pmel restriction sites

incorporated into the primers, generating plasmid pGLY9900. The DASl 3' region was then amplified using primers RCD971 (5'- ACTAGTG TAGATTTGGCCACTAACGGGTTAGTAG-3¹ ) (SEQ ID NO : 14) and RCD972 (5'- GTCGACGGCCGATGGGGC CGATGTAAGACGTGACGATGATTGG-3¹ ) (SEQ ID NO: 15). This region was cloned into the Topo TA cloning vector (Invitrogen, Carlsbad, CA) and sequenced. The DASl 3' fragment was then

subcloned into plasmid pGLY9900 using the Spel/Sail restriction sites incorporated into the primers, generating plasmid pGLY9903 (Figure 5) .

The dasl : :URA5 knockout plasmid was transformed into the ura5- P. pastoris glycoengineered strain YGLY16676. Strain YGLY16676 is a ura5- and 5-FOA resistant descendent of strain YGLY13992, which has been engineered to express genes required for human complex N- glycans with terminal galactose (Bobrowicz, 2004), as well as a secreted anti-HER2 humanized mAb (Goldenberg, 1999) . Clones were selected on synthetic medium lacking uracil to select for those clones in which the dasl : : URA5 allele has replaced the endogenous P. pastoris DASl locus by homologous recombination (Figure 4) .

Clones that lacked the DASl gene were screened by PCR using primers RCD835 (5'- CCGGG CGTTGCCATTTCTGAGTGC-3 ' ) (SEQ ID NO: 16) and

RCD1012 (5 ' -GTTTGACCAACAATCCCTGGCA-3 ' ) (SEQ ID NO: 17) and

identifying clones that lacked a PCR amplified fragment. Two clones were selected and were streak isolated and named YGLY24960 and YGLY24961. A single URA5+ but DAS1+ ectopically integrated control strain was also streak isolated and named YGLY24966. Likewise, using the DAS1/DAS2 locus depicted in Figure 4, a das2::URA5 deletion allele is constructed which, when integrated, results in a strain that only has dihydroxyacetone synthase activity from DAS1. This das2 strain is similar to the dasl deletion strain in that the reduction of DAS activity decreases flux through the assimilatory pathway and increases flux in the dissimilatory pathway as depicted in Figure 3.

Example 3 ; Production of an anti-HER2 monoclonal antibody in a Dihydroxyacetone Synthase mutant of P. pastoris under mini- bioreactor conditions

Two glycoengineered P. pastoris dasl mutant strains, YGLY24960 and YGLY24961, expressing an anti-HER2 mAb, as described above, were cultivated in a modified version of an Applikon (Foster City, CA) micro24 5ml mini-fermenter apparatus, along with the DAS1+ ectopic control YGLY24966 and the parental DAS1+ strain YGLY13992. Seed cultures were prepared by inoculating strains from YSD plates to a Whatman 24-well Uniplate (10 ml, natural polypropylene) containing 3.5 ml of 4% BMGY medium (Invitrogen, Carlsbad, CA) buffered to pH 6.0 with potassium phosphate buffer. The seed cultures were grown for approximately 65-72 hours in a temperature controlled shaker at 24°C and 650 rpm agitation. 1.0 ml of the 24 well plate grown seed culture and 4.0ml of 4% BMGY medium was then used to inoculate each well of a Micro24 plate (Type : REG2) . 30 ml of Antifoam 204 (1:25 dilution, Sigma Aldrich) was added to each well . The Micro24 was operated in Microaerobicl mode and the fermentations were controlled at 200% dissolved oxygen, pH at 6.5, temperature at 24°C and agitation at 800rpm. The induction phase was initiated upon observance of a dissolved oxygen (DO) spike after the growth phase by adding bolus shots of methanol feed solution (100% [w/w] methanol, 5 mg/1 biotin and 12.5 ml/1 PTM2 salts) , 50μ1 in the morning and 125μ1 in the afternoon. After approximately 72 hours of methanol induction, the cell-free culture supernatant was harvested by centrifugation at 2500 x g in a

Beckman swinging bucket centrifuge and subjected to protein A purification by standard methods (Jiang, 2011) . Antibody was quantified by reverse phase HPLC and calculated on a per liter basis. The two dasl mutants produced, on average, 564 mg/L of anti-HER2, or 1.7 fold more antibody than the parental control and 1.6 fold more than the ectopic DAS1+ control strain (Figure 6) .

Additionally, the culture supernatants were examined for the presence of dsDNA, indicative of cell lysis, using the Quant-iT™ PicoGreen® dsDNA Assay Kit (Invitrogen, Carlsbad, CA) . After 72 hours of methanol induction, supernatant DNA levels of the two dasl mutant strains averaged 8.2 μg/mL while the parental control (16.3 μg/mL) and the DAS1+ ectopic control (22.4 μg/mL) strains had significantly higher supernatant DNA levels (Figure 7) . This indicated that the dasl mutation not only increased the

productivity of a P. pastoris mAb-producing strain, but can also improved the strain robustness as measured by dsDNA accumulation.

Example 4 ; Production of an anti-HER2 monoclonal antibody in a dihydroxyacetone synthase mutant of P. pastoris using two

distinctly controlled industrially scalable bioprocesses

A P. pastoris dasl: :URA5 mutant strain, YGLY24961 and the parental control, YGLY13992, were each cultivated in 3 L and 15 L glass bioreactors (Applikon, Foster City, CA) . For fermentation in a 3L bioreactor, a vial (lmL) of RCB (Research Cell Bank) was inoculated into 500 mL of BSGY medium (4% glycerol, 1% yeast extract, 2% Soytone, 1.34% YNB without amino acids, 0.23% K₂HP0₄, 1.19% KH₂P0₄, 8 μg/L biotin) in a 1 L-baffled flask. The culture incubated at 24°C, while shaking on an orbital shaker at 180 rpm for 48 ± 4 hours. The bioreactor was inoculated with a 10% volumetric ratio of seed to initial BSGY medium. Cultivation conditions were the following: temperature set at 24 + 0.5°C, pH controlled at 6.5 ± 0.1 with 30% ammonium hydroxide, dissolved oxygen was maintained at 20% of saturation by cascading agitation rate on the addition of pure oxygen to the fixed airflow rate of 0.7 wm. After depletion of the initial glycerol (4%) , a 50% glycerol solution containing 12.5 mL/L of PTM1 salts (6.5g FeS0₄-7H₂0, 2. Og ZnCl₂, 0.6g

CuS0₄-5H₂0, 3.0g MnS0₄ · 7H₂0, 0.5g CoCl₂-6H₂0, 0.2g NaMo0₄-2H₂0, 0.2g biotin, 80mg Nal, 20mg H₃B0₄ per L) was fed exponentially at a rate of 0.08 h^"1 for 8h. Induction was initiated after a 30 minutes starvation phase when methanol was fed.

Anti-Her2 antibody was expressed and secreted into medium using two different conditions for feeding methanol. First, strains were cultivated in C-limited condition. Methanol was fed exponentially starting at 1.33 g/L/h increasing at a rate of 0.0063 h^"1 and the entire induction phase was conducted under methanol limited conditions. Next, strains were cultivated in DO-limited condition with excess methanol. Agitation speed was changed from cascade mode to manual mode to set 900 rpm for maintaining OTR

(Oxygen Transfer Rate) around 55 + 5 mmol/L/h and methanol was fed by a feedback control loop between methanol a sensor (Raven,

Canada) and pump to maintain methanol concentration at 3 ± 1 g/L during induction phase.

Example 5 ; Overexpression of members of the methanol

dissimilation pathway

Deletion of either DASl resulted in a strain with reduced flux through the methanol assimilatory pathway, resulting in

formaldehyde removal from the peroxisome in the form of S- hydroxymethyl glutathione, which initiates the detoxification dissimilatory pathway to convert formaldehyde into C0₂. To further increase the capacity of the dissimilatory pathway to process the excess formaldehyde, it may be further necessary to increase the expression of one or more genes in this pathway. A P. pastoris strain that is either wild type for DASl and DAS2 or has had one of the DAS genes deleted is further engineered by introduction of a plasmid containing a gene cassette encoding for FLD1 (SEQ ID NO: 7), FGH1 (SEQ ID NO: 9), or FAD1 (SEQ ID NO: 11), or all three, as in the case of pGLY-MeOHDIS (Figure 8) . Overexpression plasmids are constructed first by PCR amplification of the FLD1 gene using primers (5 ' -GAATTCATGTCTACCGAAGGTCAAGTAAGT-3¹ ) (SEQ ID NO: 18) and (5'-GGCCGGCC TTAGTGCATAGTAATCACAGCACG-3 ' ) (SEQ ID NO: 19), the FGH1 gene using primers (5'-GAA TTCATGTCATCAATTACTACTTCAATC-3¹ ) (SEQ ID NO: 20) and (5¹ -GGCCGGCCCTAGTTTAACCCCAAATA CTTTGC-3¹ ) (SEQ ID NO: 21), and the FAD2 gene using primers (5 ' -GAATTCATGAAAATCGTTC TCGTTTTGTAC-3¹ ) (SEQ ID NO: 22) and (5'-

GGCCGGCCTTATGCGACCTTTTTGTCATTACC-3¹ ) (SEQ ID NO: 23). Each amplified PCR product is then digested with £coRI/FseI and

introduced into plasmid pGLY8369 by subcloning using the introduced EcoRI/Fsel restriction sites, to generate pGLY-FLDl (Figure 9) , pGLY-FGHl (Figure 10) , and pGLY-FADl (Figure 11) . The FGH1 gene cassette is then introduced into plasmid pGLY-FLDl by digestion with Bam l/'Bglll and insertion into the unique Bglll site of pGLY- FLD1 to generate pGLY-FLDlFGHl . The FAD1 gene cassette is inserted into the pGLY-FLD1FGH1 plasmid by digestion with BamEl/Bglll and insertion into the unique Bglll site of pGLY-FLDlFGHl to generate plasmid pGLY-MeOHDIS (Figure 8) .

Plasmids pGLY-FLDl , pGLY-FGHl, pGLY-FADl, and pGLY-MeOHDIS are digested with Spel, which linearizes the plasmids to promote roll- in integration into the Pichia pastoris URA6 gene, introduced into either a DAS1/DAS2 wild type or dasl or das2 deletion strain by electroporation, and the resulting clones selected on medium containing Arsenite. The isolated clones are examined to

demonstrate increased titer of secreted protein (e.g., mAb) and improved robustness of secreted protein under bioreactor conditions as described in Examples 3 and 4.

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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.

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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.

Claims

We claim:

1. An isolated fungal host cell comprising a knocked-out DAS1 or DAS2 which comprises a heterologous polynucleotide encoding a heterologous polypeptide.

2. The cell of claim 1 which is a Pichia cell.

3. The cell of claim 2 wherein the Pichia cell is Pichia pastoris .

4. The cell of claim 1 wherein DAS1 is knocked-out.

5. The cell of claim 1 wherein DAS2 is knocked-out.

6. The cell of any of claims 1-5 that overexpresses one or more members of the cellular methanol dissimilation pathway.

7. The cell of claim 1 that overexpresses one or more members selected from the group consisting of FLD1, FGH1 and FADl .

8. The cell of claim 1 wherein the heterologous polynucleotide encodes an immunoglobulin.

9. The cell of claim 1 in a growth medium.

10. The cell of claim 1 which is genetically engineered to alter a glycosylation pattern on said heterologous polypeptide.

11. The cell of claim 1 wherein:

(i) the heterologous polynucleotide encodes a heterologous

polypeptide which is an immunoglobulin wherein said immunoglobulin forms part of an antibody or antigen-binding fragment thereof that bind specifically to VEGF, HERl, HER2, HER3, glycoprotein Ilb/lIIa, CD52, IL-2R alpha receptor (CD25) , epidermal growth factor receptor (EGFR) , Complement system protein C5, CDlla, TNF alpha, CD33, IGF1R, CD20, T cell CD3 Receptor, alpha-4 (alpha 4) integrin, PCSK9, immunoglobulin E (IgE) , RSV F protein or ErbB2. Other PCSK9, immunoglobulin E (IgE) , RSV F protein or ErbB2. Other examples of said heterologous polynucleotides encode: VEGF, HER1, HER2, HER3 , glycoprotein lib/Ilia, CD52, IL-2R alpha receptor

Receptor, alpha- 4 (alpha 4) integrin, PCSK9, immunoglobulin E

(IgE), RSV F protein or ErbB2 ; or wherein said immunoglobulin forms part of an antibody or antigen-binding fragment thereof that is Abciximab; Adalimumab; Alemtuzumab; Basiliximab; Bevacizumab;

Cetuximab; Certolizumab; Daclizumab; Dalotuzumab ; Denosumab;

Eculizumab; Efalizumab; Gemtuzumab; Ibritumomab tiuxetan;

Infliximab; uromonab-CD3 ; Natalizumab; Omalizumab; Palivizumab; Panitumumab; Ranibizumab; Rituximab; Tositumomab; or Trastuzumab.

(ii) the DASl polynucleotide comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence set forth in

SEQ ID NO: 2 ;

(iii) the DAS2 polynucleotide comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence set forth in SEQ ID NO: 3 ;

(iv) the DASl polypeptide comprises an amino acid sequence that is at least 80% identical or similar to the amino acid sequence set forth in SEQ ID NO: 4 ;

(v) the DAS2 polypeptide comprises an amino acid sequence that is at least 80% identical or similar to the amino acid sequence set forth in SEQ ID NO: 5;

(vi) the heterologous polynucleotide is operably linked to a methanol -inducible promoter; or

(vii) said cell produces glycoproteins comprising

GlcNAc₂Man₃GlcNAc₂.

12. A method for producing an isolated fungal host cell which comprises a heterologous polynucleotide encoding a heterologous polypeptide comprising knocking out chromosomal DASl or DAS2 in a fungal cell and introducing said polynucleotide into the cell.

13. The method of claim 12 wherein DASl or DAS2 is knocked out by interrupting, point mutating or deleting chromosomal DASl or DAS2 in the fungal cell.

14. The method of claim 12 wherein the fungal cell is Pichia pastoris and DASl is knocked-out by interrupting chromosomal DASl by causing genetic recombination of a Pichia pastoris URA5 gene at the DASl chromosomal locus as described in Figure 4.

15. A method for producing a heterologous polypeptide comprising introducing a heterologous polynucleotide encoding said polypeptide into an isolated fungal cell in which DASl or DAS2 is knocked-out and culturing said cell under conditions where the heterologous polypeptide is expressed in said cell.

16. The method of claim 15 wherein said heterologous polypeptide is an immunoglobulin.

17. The method of claim 15 wherein the heterologous polypeptide is secreted from the host cell.

18. The method of claim 15 further comprising purifying the polypeptide .