Description PICHIA METHANOLICA GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE 2 PROMOTER AND TERMINATOR BACKGROUND OF THE INVENTION Methylotrophic yeasts are those yeasts that are able to utilize methanol as a sole source of carbon and energy. Species of yeasts that have the biochemical pathways necessary for methanol utilization are classified in four genera, Hansenula, Pichia, Candida, and Torulopsis. These genera are somewhat artificial, having been based on cell morphology and growth characteristics, and do not reflect close genetic relationships (Billon-Grand, Mycotaxon 35: 201-204,1989; Kurtzman, Mycologia 84: 72-76,1992). Furthermore, not all species within these genera are capable of utilizing methanol as a source of carbon and energy. As a consequence of this classification, there are great differences in physiology and metabolism between individual species of a genus.

Methylotrophic yeasts are attractive candidates for use in recombinant protein production systems for several reasons. First, some methylotrophic yeasts have been shown to grow rapidly to high biomass on minimal defined media. Second, recombinant expression cassettes are genomically integrated and therefore mitotically stable. Third, these yeasts are capable of secreting large amounts of recombinant proteins. See, for example, Faber et al., Yeast 11: 1331,1995; Romanos et al., Yeast 8: 423,1992; Cregg et al., Bio/Technolog 11: 905,1993; U. S. Patent No. 4,855,242; U. S. Patent No. 4,857,467; U. S. Patent No. and U. S. Patent No. 4,929,555; and Raymond, U. S. Patents Nos. 5,716,808,5,736,383,5,854,039, and 5,888,768.

Previously described expression systems for methylotrophic yeasts rely largely on the use of methanol-inducible transcription promoters. The use of methanol- induced promoters is, however, problematic as production is scaled up to commercial levels. The overall volume of methanol used during the fermentation process can be as much as 40% of the final fermentation volue, and at 1000-liter fermentation scale and above the volumes of methanol required for induction necessitate complex and potentially expensive considerations.

There remains a need in the art for additional materials and methods to enable the use of methylotrophic yeasts for production of polypeptides of economic

importance, including industrial enzymes and pharmaceutical proteins. The present invention provides such materials and methods as well as other, related advantages.

SUMMARY OF THE INVENTION It is an object of the present invention to provide transcription promoter and terminator sequences for use in Pichia methanolica. It is a further object of the invention to provide materials and methods for obtaining constitutive expression of heterologous DNA in P. methanolica. It is also an object of the invention to provide methods for production of polypeptides in P. methanolica, which methods can be readily scaled up to industrial levels, and to provide materials that can be used within these methods. It is another object of the invention to provide materials and methods for obtaining constitutive transcription of heterologous DNA to produce recombinant proteins in P. methanolica.

Within one aspect, the present invention provides an isolated DNA molecule of up to 5000 nucleotides in length comprising nucleotide 93 to nucleotide 1080 of SEQ ID NO: 1.

Within a second aspect of the invention there is provided a DNA construct comprising the following operably linked elements: a first DNA segment comprising at least a portion of the sequence of SEQ ID NO: 1 from nucleotide 93 to nucleotide 1092, wherein the portion is a functional transcription promoter; a second DNA segment encoding a protein of interest other than a Pichia methanolica glyceraldehyde-3-phosphate dehydrogenase; and a third DNA segment comprising a transcription terminator. Within one embodiment, the first DNA segment is from 900 to 1500 nucleotides in length. Within another embodiment, the first DNA segment is from 900 to 1000 nucleotides in length. Within an additional embodiment, the first DNA segment is substantially free of Pichia methanolica glyceraldehyde-3-phosphate dehydrogenase gene coding sequence. The DNA construct may further comprise a selectable marker, preferably a Pichia methanolica gene, more preferably a Pichia methanolica ADE2 gene. The DNA construct may be a closed, circular molecule or a linear molecule. Within other embodiments, the DNA constuct further comprises a secretory signal sequence, such as the S. cerevisiae alpha-factor pre-pro sequence, operably linked to the first and second DNA segments. Within additional embodiments, the third DNA segment comprises a transcription terminator of a Pichia methanolica A UG1 or GAP2 gene.

Within a third aspect of the invention there is provided a Pichia methanolica cell containing a DNA construct as disclosed above. Within one embodiment, the DNA construct is genomically integrated. Within a related

embodiment, the DNA construct is genomically integrated in multiple copies. Within a further embodiment, the P. methanolica cell is functionally deficient in vacuolar proteases proteinase A and proteinase B.

Within a fourth aspect of the invention there is provided a method of producing a protein of interest comprising the steps of (a) culturing a P. methanolica cell as disclosed above whereby the second DNA segment is expressed and the protein of interest is produced, and (b) recovering the protein of interest from the cultured cell.

Within a fifth aspect of the invention there is provided a DNA construct comprising the following operably linked elements: a first DNA segment comprising a Pichia methanolica gene transcription promoter; a second DNA segment encoding a protein of interest other than a Pichia methanolica protein; and a third DNA segment comprising nucleotides 2095 to 2145 of SEQ ID NO: 1.

These and other aspects of the invention will become evident upon reference to the following detailed description of the invention.

DETAILED DESCRIPTION OF THE INVENTION The term"allelic variant"is used herein to denote an alternative form of a gene. Allelic variation is known to exist in populations and arises through mutation.

A"DNA construct"is a DNA molecule, either single-or double- stranded, that has been modified through human intervention to contain segments of DNA combined and juxtaposed in an arrangement not existing in nature.

A"DNA segment"is a portion of a larger DNA molecule having specified attributes. For example, a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, that, when read from the 5'to the 3'direction, encodes the sequence of amino acids of the specified polypeptide.

The term"functionally deficient"denotes the expression in a cell of less than 10% of an activity as compared to the level of that activity in a wild-type counterpart. It is preferred that the expression level be less than 1 of the activity in the wild-type counterpart, more preferably less than 0.01% as determined by appropriate assays. It is most preferred that the activity be essentially undetectable (i. e., not significantly above background). Functional deficiencies in genes can be generated by mutations in either coding or non-coding regions.

The term"gene"is used herein to denote a DNA segment encoding a polypeptide. Where the context allows, the term includes genomic DNA (with or without intervening sequences), cDNA, and synthetic DNA. Genes may include non- coding sequences, including promoter elements.

The term"isolated", when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.

"Operably linked", when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e. g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.

A"polynucleotide"is a single-or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5'to the 3'end.

Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules.

Sizes of polynucleotides are expressed as base pairs (abbreviated"bp"), nucleotides ("nt"), or kilobases ("kb"). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double-stranded. When these terms are applied to double-stranded molecules they are used to denote overall length and will be understood to be equivalent to the term"base pairs". It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length.

A"polypeptide"is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as"peptides".

The term"promoter"is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5'non-coding regions of genes. Sequences within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. These promoter elements include RNA polymerase binding sites, TATA sequences, and transcription factor binding sites. See, in general, Watson et al., eds., Molecular Biology of the Gene, 4th ed., The Benjamin/Cummings Publishing Company, Inc., Menlo Park, CA, 1987.

A"pro sequence"is a DNA sequence that commonly occurs immediately 5'to the mature coding sequence of a gene encoding a secretory protein.

The pro sequence encodes a pro peptide that serves as a cis-acting chaperone as the protein moves through the secretory pathway.

A"protein"is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell.

Proteins are commonly defined in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.

The term"secretory signal sequence"denotes a DNA sequence that encodes a polypeptide (a"secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway. A secretory peptide and a pro peptide may be collectively referred to as a pre-pro peptide.

The present invention provides isolated DNA molecules comprising a Pichia methanolica glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene promoter. The invention also provides isolated DNA molecules comprising a P. methanolica GAPDH gene terminator. The promoter and terminator can be used within methods of producing proteins of interest, including proteins of pharmaceutical or industrial value.

The sequence of a DNA molecule comprising a Pichia methanolica glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene promoter, coding region, and terminator is shown in SEQ ID NO: 1. The gene has been designated GAP2. Those skilled in the art will recognize that SEQ ID NO: 1 represents a single allele of the P. methanolica GAP2 gene and that other functional alleles (allelic variants) are likely to exist, and that allelic variation may include nucleotide changes in the promoter region, coding region, or terminator region.

The partial sequence of a second P. methanolica glyceraldehyde-3- phosphate dehydrogenase gene, designated GAP1, is shown in SEQ ID NO: 2.

Within SEQ ID NO: 1, the GAPDH open reading frame begins with the methionine codon (ATG) at nucleotides 1093-1095. The transcription promoter is located upstream of the ATG. Gene expression experiments showed that a functional promoter was contained within the ca. 1000 nucleotide 5'-flanking region of the GAP2 gene.

Preferred portions of the sequence shown in SEQ ID NO: 1 for use within the present invention as transcription promoters include segments comprising at

least 900 contiguous nucleotides of the 5'non-coding region of SEQ ID NO: 1, and preferably comprising nucleotide 93 to nucleotide 1080 of the sequence shown in SEQ ID NO: 1. Those skilled in the art will recognize that longer portions of the 5'non- coding region of the P. methanolica GAP2 gene can also be used. Promoter sequences of the present invention can thus include the sequence of SEQ ID NO: 1 through nucleotide 1092 in the 3'direction and can extend to or beyond nucleotide 1 in the 5' direction. In general, the promoter used within an expression DNA construct will not exceed 1.5 kb in length, and will preferably not exceed 1.0 kb in length. In addition to these promoter fragments, the invention also provides isolated DNA molecules of up to about 3300 bp, as well as isolated DNA molecules of up to 5000 bp, wherein said molecules comprise the P. methanolica GAP2 promoter sequence.

As disclosed in more detail in the examples that follow, the sequence of SEQ ID NO: 1 from nucleotide 93 to nucleotide 1080 provides a functional transcription promoter. However, additional nucleotides can be removed from either or both ends of this sequence and the resulting sequence tested for promoter function by joining it to a sequence encoding a protein, preferably a protein for which a convenient assay is readily available.

Within the present invention it is preferred that the GAP2 promoter be substantially free of GAP2 gene coding sequence, which begins with nucleotide 1093 in SEQ ID NO: 1. As used herein,"substantially free"of GAP2 gene coding sequence means that the promoter DNA includes not more than 15 nucleotides of the GAP2 coding sequence, preferably not more than 10 nucleotides, and more preferably not more than 3 nucleotides. Within a preferred embodiment of the invention, the GAP2 promoter is provided free of coding sequence of the P. methanolica GAP2 gene.

However, those skilled in the art will recognize that a GAP2 gene fragment that includes the initiation ATG (nucleotides 1093 to 1095) of SEQ ID NO: 1 can be operably linked to a heterologous coding sequence that lacks an ATG, with the GAP2 ATG providing for intition of translation of the heterologous sequence. Those skilled in the art will further recognize that additional GAP2 coding sequences can also be included, whereby a fusion protein comprising GAP2 and heterologous amino acid sequences is produced. Such a fusion protein may comprise a cleavage site to facilitate separation of the GAP2 and heterologous sequences subsequent to translation.

In addition to the GAP2 promoter sequence, the present invention also provides transcription terminator sequences derived from the 3'non-coding region of the P. methanolica GAP2 gene. A consensus transcription termination sequence (Chen and Moore, Mol. Cell. Biol. 12: 3470-3481,1992) is at nucleotides 2136 to 2145 of SEQ ID NO: 1. Within the present invention, there are thus provided transcription terminator

gene segments of at least about 50 bp, preferably at least 60 bp, more preferably at least 90 bp, still more preferably about 200 bp in length. The terminator segments of the present invention may comprise 500-1000 nucleotides of the 3'non-coding region of SEQ ID NO: 1. These segments comprise the termination sequence disclosed above, and preferably have as their 5'termini nucleotide 2095 of SEQ ID NO: 1. Those skilled in the art will recognize, however, that the transcription terminator segment that is provided in an expression vector can include at its 5'terminus the TAA translation termination codon at nucleotides 2092-2094 of SEQ ID NO: 1 to permit the insertion of coding sequences that lack a termination codon.

Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are well known in the art and are disclosed by, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Murray, ed., Gene Transfer and Expression Protocols, Humana Press, Clifton, NJ, 1991; Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D. C., 1994; Ausubel et al. (eds.), Short Protocols in Molecular Biology, 3rd edition, John Wiley and Sons, Inc., NY, 1995; Wu et al., Methods in Gene Biotechnology, CRC Press, New York, 1997. DNA vectors, including expression vectors, commonly contain a selectable marker and origin of replication that function in a bacterial host (e. g., E. coli) to permit the replication and amplification of the vector in a prokaryotic host. If desired, these prokaryotic elements can be removed from a vector before it is introduced into an alternative host. For example, such prokaryotic sequences can be removed by linearization of the vector prior to its introduction into a P. methanolica host cell.

Within certain embodiments of the invention, expression vectors are provided that comprise a first DNA segment comprising at least a portion of the sequence of SEQ ID NO: 1 that is a functional transcription promoter operably linked to a second DNA segment encoding a protein of interest. When it is desired to secrete the protein of interest, the vector will further comprise a secretory signal sequence operably linked to the first and second DNA segments. The secretory signal sequence may be that of the protein of interest, or may be derived from another secreted protein, preferably a secreted yeast protein. A preferred such yeast secretory signal sequence is the S. cerevisiae alpha-factor (MFal) pre-pro sequence (disclosed by Kurjan et al., U. S.

Patent No. 4,546,082 and Brake, U. S. Patent No. 4,870,008).

Within other embodiments of the invention, expression vectors are provided that comprise a DNA segment comprising a portion of SEQ ID NO: 1 that is a functional transcription terminator operably linked to an additional DNA segment

encoding a protein of interest. Within one embodiment, the GAP2 promoter and terminator sequences of the present invention are used in combination, wherein both are operably linked to a DNA segment encoding a protein of interest within an expression vector.

Expression vectors of the present invention further comprise a selectable marker to permit identification and selection of P. methanolica cells containing the vector. Selectable markers provide for a growth advantage of cells containing them.

The general principles of selection are well known in the art. The selectable marker is preferably a P. methanolica gene. Commonly used selectable markers are genes that encode enzymes required for the synthesis of amino acids or nucleotides. Cells having mutations in these genes cannot grow in media lacking the specific amino acid or nucleotide unless the mutation is complemented by the selectable marker. Use of such "selective"culture media ensures the stable maintenance of the heterologous DNA within the host cell. A preferred selectable marker of this type for use in P. methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5- aminoimidazole carboxylase (AIRC; EC 4.1.1.21). See, Raymond, U. S. Patent No.

5,736,383. The ADE2 gene, when transformed into an ade2 host cell, allows the cell to grow in the absence of adenine. The coding strand of a representative P. methanolica ADE2 gene sequence is shown in SEQ ID NO: 3. The sequence illustrated includes 1006 nucleotides of 5'non-coding sequence and 442 nucleotides of 3'non-coding sequence, with the initiation ATG codon at nucleotides 1007-1009. Within a preferred embodiment of the invention, a DNA segment comprising nucleotides 407-2851 is used as a selectable marker, although longer or shorter segments could be used as long as the coding portion is operably linked to promoter and terminator sequences. In the alternative, a dominant selectable marker, which provides a growth advantage to wild- type cells, may be used. Typical dominant selectable markers are genes that provide resistance to antibiotics, such as neomycin-type antibiotics (e. g., G418), hygromycin B, and bleomycin/phleomycin-type antibiotics (e. g., ZeocinTM; available from Invitrogen Corporation, San Diego, CA). A preferred dominant selectable marker for use in P. methanolica is the Sh bla gene, which inhibits the activity of ZeocinTM.

The use of P. methanolica cells as a host for the production of recombinant proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565; and U. S. Patents Nos. 5,716,808,5,736,383, 5,854,039, and 5,736,383. Expression vectors for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation. To facilitate integration of the expression vector DNA into the host chromosome, it is preferred to have the entire expression segment of

the plasmid flanked at both ends by host DNA sequences (e. g., At/G7 3'sequences).

Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P. methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (T) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.

Integrative transformants are preferred for use in protein production processes. Such cells can be propagated without continuous selective pressure because DNA is rarely lost from the genome. Integration of DNA into the host chromosome can be confirmed by Southern blot analysis. Briefly, transformed and untransformed host DNA is digested with restriction endonucleases, separated by electrophoresis, blotted to a support membrane, and probed with appropriate host DNA segments. Differences in the patterns of fragments seen in untransformed and transformed cells are indicative of integrative transformation. Restriction enzymes and probes can be selected to identify transforming DNA segments (e. g., promoter, terminator, heterologous DNA, and selectable marker sequences) from among the genomic fragments.

Differences in expression levels of heterologous proteins can result from such factors as the site of integration and copy number of the expression cassette among individual isolates. It is therefore advantageous to screen a number of isolates for expression level prior to selecting a production strain. Isolates exhibiting a high expression level will commonly contain multiple integrated copies of the desired expression cassette. A variety of suitable screening methods are available. For example, transformant colonies are grown on plates that are overlayed with membranes (e. g., nitrocellulose) that bind protein. Proteins are released from the cells by secretion or following lysis, and bind to the membrane. Bound protein can then be assayed using known methods, including immunoassays. More accurate analysis of expression levels can be obtained by culturing cells in liquid media and analyzing conditioned media or cell lysates, as appropriate. Methods for concentrating and purifying proteins from media and lysates will be determined in part by the protein of interest. Such methods are readily selected and practiced by the skilled practitioner.

For production of secreted proteins, host cells having functional deficiencies in the vacuolar proteases proteinase A, which is encoded by the PEP4 gene, and proteinase B, which is encoded by the PRBI gene, are preferred in order to minimize spurious proteolysis. Vacuolar protease activity (and therefore vacuolar protease deficiency) is measured using any of several known assays. Preferred assays are those developed for Saccharomyces cerevisiae and disclosed by Jones, Methods

Enzymol. 194: 428-453,1991. A preferred such assay is the APNE overlay assay, which detects activity of carboxypeptidase Y (CpY). See, Wolf and Fink, J. Bact. 123: 1150- 1156,1975. Because the zymogen (pro) CpY is activated by proteinase A and proteinase B, the APNE assay is indicative of vacuolar protease activity in general. The APNE overlay assay detects the carboxypeptidase Y-mediated release of p-naphthol from N-acetyl-phenylalanine-p-naphthyl-ester (APNE), which results in the formation of an isoluble red dye by the reaction of the-naphthol with the diazonium salt Fast Garnet GBC. Cells growing on assay plates (YEPD plates are preferred) at room temperature are overlayed with 8 ml RxM. RxM is prepared by combining 0.175 g agar, 17.5 ml HO, and 5 ml 1 M Tris-HCl pH 7.4, microwaving the mixture to dissolve the agar, cooling to-55°C, adding 2.5 ml freshly made APNE (2 mg/ml in dimethylformamide) (Sigma Chemical Co., St. Louis, MO), and, immediately before assay, 20 mg Fast Garnet GBC salt (Sigma Chemical Co.). The overlay is allowed to solidify, and color development is observed. Wild-type colonies are red, whereas CPY deletion strains are white. Carboxypeptidase Y activity can also be detected by the well test, in which cells are distributed into wells of a microtiter test plate and incubated in the presence of N-benzoyl-L-tyrosine p-nitroanilide (BTPNA) and dimethylformamide.

The cells are permeabilized by the dimethylformamide, and CpY in the cells cleaves the amide bond in the BTPNA to give the yellow product p-nitroaniline. Assays for CpY will detect any mutation that reduces protease activity so long as that activity ultimately results in the reduction of CpY activity.

P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25°C to 35°C.

Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors. A preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% BactoTM Peptone (Difco Laboratories, Detroit, MI), 1% Bacto yeast extract (Difco Laboratories), 0.004% adenine, 0.006% L-leucine).

For large-scale culture, one to two colonies of a P. methanolica strain can be picked from a fresh agar plate (e. g, YEPD agar) and suspended in 250 ml of YEPD broth contained in a two-liter baffled shake flask. The culture is grown for 16 to 24 hours at 30°C and 250 rpm shaking speed. Approximately 50 to 80 milliliters of inoculum are used per liter starting fermentor volume (5-8% v/v inoculum).

A preferred fermentation medium is a soluble medium comprising glucose as a carbon source, inorganic ammonia, potassium, phosphate, iron, and citric acid. As used herein, a"soluble medium"is a medium that does not contain visible precipitation. Preferably, the medium lacks phosphate glass (sodium

hexametaphosphate). A preferred medium is prepared in deionized water and does not contain calcium sulfate. As a minimal medium, it is preferred that the medium lacks polypeptides or peptides, such as yeast extracts. However, acid hydrolyzed casein (e. g., casamino acids or amicase) can be added to the medium if desired. An illustrative fermentation medium is prepared by mixing the following compounds: (NH4) 2SO4 (11.5 grams/liter), K2HP04 (2.60 grams/liter), KH,) P04 (9.50 grams/liter), FeS04*7H20 (0.40 grams/liter), and citric acid (1.00 gram/liter). After adding distilled, deionized water to one liter, the solution is sterilized by autoclaving, allowed to cool, and then supplemented with the following: 60% (w/v) glucose solution (47.5 milliliters/liter), lOx trace metals solution (20.0 milliliters/liter), 1 M MgS04 (20.0 milliliters/liter), and vitamin stock solution (2.00 milliliters/liter). The lOx trace metals solution contains FeSO4*7H2O (100 mM), CuS045H20 (2 mM), ZnSO4*7H2O (8 mM), MnS04. H20 (8 mM), CoCl2*6H20 (2 mM), Na2Mo04'2H20 (1 mM), H3BO3 (8 mM), KI (0.5 mM), NiSO4. 6H2O (1 mM), thiamine (0.50 grams/liter), and biotin (5.00 milligrams/liter).

The vitamin stock solution contains inositol (47.00 grams/liter), pantothenic acid (23.00 grams/liter), pyrodoxine (1.20 grams/liter), thiamine (5.00 grams/liter), and biotin (0.10 gram/liter). Those of skill in the art can vary these particular ingredients and amounts.

For example, ammonium sulfate can be substituted with ammonium chloride, or the amount of ammonium sulfate can be varied, for example, from about 11 to about 22 grams/liter.

After addition of trace metals and vitamins, the pH of the medium is typically adjusted to pH 4.5 by addition of 10% H3PO4. Generally, about 10 milliliters/liter are added, and no additional acid addition will be required. During fermentation, the pH is maintained between about 3.5 to about 5.5, or about 4.0 to about 5.0, depending on protein produced, by addition of 5 N NH40H.

An illustrative fermentor is a BIOFLO 3000 fermentor system (New Brunswick Scientific Company, Inc.; Edison, NJ). This fermentor system can handle either a six-liter or a fourteen-liter fermentor vessel. Fermentations performed with the six-liter vessel are prepared with three liters of medium, whereas fermentations performed with the fourteen-liter vessel are prepared with six liters of medium. The fermentor vessel operating temperature is typically set to 30°C for the course of the fermentation, although the temperature can range between 27-31°C depending on the protein expressed. The fermentation is initiated in a batch mode. The glucose initially present is often used by approximately 10 hours elapsed fermentation time (EFT), at which time a glucose feed can be initiated to increase the cell mass. An illustrative glucose feed contains 900 milliliters of 60% (w/v) glucose, 60 milliliters of 50% (w/v) (NH4) 2SO4,60 milliliters of lOx trace metals solution, and 30 milliliters of 1 M MgS04.

Pichia methanolica fermentation is robust and requires high agitation, aeration, and oxygen sparging to maintain the percentage dissolved oxygen saturation above 30%.

The percentage dissolved oxygen should not drop below 15% for optimal expression and growth. The biomass typically reaches about 30 to about 80 grams dry cell weight per liter at 48 hours EFT.

Proteins produced according to the present invention are recovered from the host cells using conventional methods. If the protein is produced intracellulary, the cells are harvested (e. g., by centrifugation) and lysed to release the cytoplasmic contents. Methods of lysis include enzymatic and mechanical disruption. The crude extract is then fractionated according to known methods, the specifics of which will be determined for the particular protein of interest. Secreted proteins are recovered from the conditioned culture medium using standard methods, also selected for the particular protein. See, in general, Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York, 1994.

The materials and methods of the present invention can be used to produce proteins of research, industrial, or pharmaceutical interest. Such proteins include enzymes, such as lipases, cellulases, and proteases; enzyme inhibitors, including protease inhibitors; growth factors such as platelet derived growth factor (PDGF), fibroblast growth factors (FGF), epidermal growth factor (EGF), vascular endothelial growth factors (VEGFs); glutamic acid decarboxylase (GAD); cytokines, such as erythropoietin, thrombopoietin, colony stimulating factors, interleukins, and interleukin antagonist; hormones, such as insulin, proinsulin, leptin, and glucagon; and receptors, including growth factor receptors, which can be expressed in truncated form ("soluble receptors") or as fusion proteins with, for example, immunoglobulin constant region sequences. DNAs encoding these and other proteins are known in the art. See, for example, U. S. Patents Nos. 4,889,919; 5,219,759; 4,868,119; 4,968,607; 4,599,311; 4,784,950; 5,792,850; 5,827,734; 4,703,008; 4,431,740; and 4,762,791; and WIPO Publications WO 95/21920 and WO 96/22308.

It is particularly preferred to use the present invention to produce unglycosylated pharmaceutical proteins. Yeast cells, including P. methanolica cells, produce glycoproteins with carbohydrate chains that differ from their mammalian counterparts. Mammalian glycoproteins produced in yeast cells may therefore be regarded as"foreign"when introduced into a mammal, and may exhibit, for example, different pharmacokinetics than their naturally glycosylated counterparts.

The invention is further illustrated by the following, non-limiting examples.

EXAMPLES Example 1 To clone the P. methanolica GAPl gene, sense (ZC11,356; SEQ ID NO: 4) and antisense (ZC11,357; SEQ ID NO: 5) PCR primers were designed from an alignment of the coding regions of GAPDH genes of Saccharomyces cerevisiae, Kluyveromyces lactis, and mouse. The primers were then used to amplify P. methanolica genomic DNA. An amplified sequence 608 bp long was recovered and was found to have 78.1% homology to the corresponding S. cerevisiae GAPDH gene sequence.

A P. methanolica genomic library was constructed in the vector pRS426 (Christianson et al., Gene 110: 119-122,1992), a shuttle vector comprising zut and S. cerevisiae URA3 sequences, allowing it to be propagated in S. cerevisiae. Genomic DNA was prepared from strain CBS6515 according to standard procedures. Briefly, cells were cultured overnight in rich media, spheroplasted with zymolyase, and lysed with SDS. DNA was precipitated from the lysate with ethanol and extracted with a phenol/chloroform mixture, then precipitated with ammonium acetate and ethanol. Gel electrophoresis of the DNA preparation showed the presence of intact, high molecular weight DNA and appreciable quantities of RNA. The DNA was partially digested with Sau 3A by incubating the DNA in the presence of a dilution series of the enzyme.

Samples of the digests were analyzed by electrophoresis to determine the size distribution of fragments. DNA migrating between 4 and 12 kb was cut from the gel and extracted from the gel slice. The size-fractionated DNA was then ligated to pRS426 that had been digested with Bam HI and treated with alkaline phosphatase.

Aliquots of the reaction mixture were electroporated into E. coli MC1061 cells using an electroporator (Gene Pulser; BioRad Laboratories, Hercules, CA) as recommended by the manufacturer.

The library was screened by PCR using sense (ZC11,733; SEQ ID NO: 6) and antisense (ZC11,734; SEQ ID NO: 7) primers designed from the sequenced region of the P. methanolica GAPDH. The PCR reaction mixture was incubated for one minute at 94°C; followed by 34 cycles of 94°C, one minute, 52°C, 45 seconds, 72°C, two minutes; and a termination cycle of 94°C, one minute, 54°C, one minute, 72°C, eleven minutes. Starting with 43 library pools, positive pools were identified and broken down to individual colonies. A single colony with a pRS426 plasmid containing the P. methanolica GAPDH gene as its insert was isolated. The orientation of the GAPDH gene and the length of the 5'and 3'flanking sequences in the insert were deduced by DNA sequencing (SEQ ID NO: 2). This gene was designated GAP7.

Within SEQ ID NO: 2, the GAPDH open reading frame begins with the methionine codon (ATG) at nucleotides 1733-1735. The transcription promoter is located upstream of the ATG. Gene expression experiments showed that a functional promoter was contained within the ca. 900 nucleotide 5'-flanking region of the GAP7 gene. Analysis of this promoter sequence revealed the presence of a number of sequences homologous to Saccharomyces cerevisiae promoter elements. These sequences include a concensus TATAAA box at nucleotides 1584 to 1591, a consensus Raplp binding site (Graham and Chambers, Nuc. Acids Res. 22: 124-130,1994) at nucleotides 1355 to 1367, and potential Gcrlp binding sites (Shore, Trends Genet.

10: 408-412,1994) at nucleotides 1225 to 1229,1286 to 1290,1295 to 1299,1313 to 1317,1351 to 1354,1370 to 1374,1389 to 1393, and 1457 to 1461. A consensus transcription termination sequence (Chen and Moore, Mol. Cell. Biol. 12: 3470-3481, 1992) was identified at nucleotides 2774 to 2787 of SEQ ID NO: 2.

A plasmid containing the GAP7 gene, designated pGAPDH, has been deposited as an E. coli strain MC1061 transformant with American Type Culture Collection, Manassas, VA under the terms of the Budapest Treaty. The deposited strain has been assigned the designation PTA-3 and a deposit date of May 4,1999.

Example 2 Analysis of the P. methanolica genome by Southern blotting, using a PCR product from the coding region of the cloned GAP7 gene as a probe, indicated the presence of three independent GAPDH genes. Primers designed from the cloned sequence were used in various combinations to amplify P. methanolica genomic DNA.

Positive pools were screened by PCR, and positives were re-amplified. PCR products were sequenced. Eight pools were found to be the same and corresponded to the previously cloned GAP7 gene. Two pools were distinct from the previously cloned gene and were identical to each other. Each of these two pools was plated and amplified by PCR through several rounds of sub-dividing. Sub-pools were streaked, and single colonies were picked for a final round of PCR screening. Positive clones were analyzed by PCR and restriction digestion. Each clone was found to be carried on a-5 kb genomic segment. This gene, which was designated GAP2, was partially sequenced. The sequenced region included an open reading frame of 1002 base pairs (including the termination codon), a 5'non-coding region of 1092 base pairs, and a 3' non-coding region of 1239 base pairs (SEQ ID NO: 1).

Example 3 A fragment of GAP2 DNA (SEQ ID NO: 1) was isolated by PCR using two primers. Primer ZC19,334 (SEQ ID NO: 8) contained 26 bp of vector flanking sequence and 25 bp corresponding to the 5'end of the first 1000 bp of the GAP2 promoter. Primer ZC19,333 (SEQ ID NO: 9) contained 35 bp of the 3'end corresponding to S. cerevisiae alpha factor pre-pro sequence and 29 bp corresponding to the 3'end of the GAP2 promoter. The latter primer altered the 5'flanking sequence at nucleotides 1081-1092 to GAATTCAAAAGA (SEQ ID NO: 10), resulting in the introduction of an EcoRI site. The PCR reaction conditions (five tubes in all) were: 20 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute; followed by a 4°C soak. The five samples were combined into one tube and precipitated with 2 volumes of 100% ethanol. The resulting pellet was resuspended in 10 ul of water. The sample was serially diluted into TE (10 mM Tris, 2mM EDTA) as 1: 5,1: 25, and 1: 125 dilutions. DNA concentration was estimated by running the PCR product on a 1% agarose gel. The expected approximately 1 kb fragment was seen. The remaining 8 Fl of product was used for recombination as described below.

An expression plasmid named pTAP96, containing the P. methanolica GAP2 promoter, S. cerevisiae alpha factor pre-pro sequence, and a cDNA encoding leptin with an amino-terminal Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl.

Acad. Sci. USA 82: 7952-4,1985), was constructed via homologous recombination using portions of the plasmids pTAP37 and pCZR189. Plasmid pTAP37 comprises a modified P. methanolica GAP7 promoter, the P. methanolica ADE2 selectable marker, the gene for ampicillin resistance in E. coli, the S. cerevisiae URA3 selectable marker, and the CEN-ARS of S. cerevisiae. pCZR189 comprises the S. cerevisiae alpha factor pre-pro sequence and the leptin coding sequence. One hundred microliters of competent yeast cells (S. cerevisiae) were combined with 7 1 of a mixture containing approximately 1 g of NotI-cut pCZR189,1 Rg PCR product containing the GAP2 promoter as described above, and 100 ng of EcoRI-cut pTAP37 vector, and the mixture was transferred to a 0.2 cm electroporation cuvette. The yeast/DNA mixture was electropulsed at 0.75 kV (5 kV/cm), infinite ohms, 25 ßF. To each cuvette was added 600 ul of 1.2 M sorbitol, and the yeast was then plated in two 300-l aliquots onto two -URA D plates and incubated at 30°C.

After about 48 hours, the Ura+ yeast transformants from a single plate were resuspended in 1 ml H20 and spun briefly to pellet the yeast cells. The cell pellet was resuspended in 1 ml of lysis buffer (2% t-octylphenoxypolyethoxyethanol (Triton0 X-100), 1% SDS, 100 mM NaCI, 10 mM Tris, pH 8.0,1 mM EDTA). Five hundred microliters of the lysis mixture was added to a microcentrifuge tube containing 300 1

acid-washed glass beads and 200 1 phenol-chloroform, vortexed for 1 minute intervals two or three times, followed by a 5 minute spin in a microcentrifuge at maximum speed. Three hundred microliters of the aqueous phase was transferred to a fresh tube, and the DNA was precipitated with 600 ul ethanol, followed by centrifugation for 10 minutes at 4°C. The DNA pellet was resuspended in 100 1 HO.

Forty 1 of electrocompetent E. coli cells (MC1061; Casadaban et al., J.

Mol. Biol. 138,179-207,1980) were transformed by electroporation with 1 jn. 1 of the yeast DNA preparation at 2.0 kV, 25 iF, and 400 ohms. Following electroporation, 0.6 ml SOC (2% Bacto Tryptone (Difco Laboratories), 0.5% yeast extract (Difco Laboratories), 10 mM NaCI, 2.5 mM KC1,10 mM MgCl2,10 mM MgS04,20 mM glucose) was plated in one aliquot on LB + Amp plates (LB broth, 1.8% Bacto Agar (Difco Laboratories), 100 mg/L Ampicillin).

Cells harboring the correct expression construct for the GAP2 promoter driving synthesis of the alpha factor pre-pro/leptin fusion were screened via PCR using the same primers used to generate the GAP2 promoter. The PCR conditions were: 25 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute; followed by a 4°C soak. Two positive clones were identified on a 1% agarose gel and were subjected to sequence analysis. One of the correct clones was selected and designated pTAP96.

Plasmid pTAP96 DNA was prepared by anion exchange chromatography using a commercially available plasmid isolation kit (QIAGENO Plasmid Maxi Kit; Qiagen, Inc., Valencia, CA). DNA was diagnostically cut with ScaI, producing the expected bands of approximately 1700 bp, 2250 bp doublet, and 6000 bp on a 1% gel. 1 Rg of pTAP96 DNA was then cut with NotI and transformed into electrocompetent P. methanolica strain PMAD16 (disclosed in Example 4, below) as disclosed in U. S. Patent No. 5,854,039. Transformants were selected on-ADE DS plates (Table 1).

Table 1 -ADE DS 0.056%-Ade-Trp-Thr powder 0.67% yeast nitrogen base without amino acids 2% D-glucose 0.5% 200X tryptophan, threonine solution 18.22% D-sorbitol -Ade-Trp-Thr powder powder made by combining 3.0 g arginine, 5.0 g aspartic acid, 2.0 g histidine, 6.0 g isoleucine, 8.0 g leucine, 4.0 g lysine, 2.0 g methionine, 6.0 g phenylalanine, 5.0 g serine, 5.0 g tyrosine, 4.0 g uracil, and 6.0 g valine (all L- amino acids) 200X tryptophan, threonine solution 3.0% L-threonine, 0.8% L-tryptophan in HO For plates, add 1.8% Bacto agar (Difco Laboratories) White colonies, indicating the presence of the ADE2 gene, were patched onto-ADE plates, and cells were allowed to grow overnight. The cells were then replica plated onto YEPD plates and overlaid with a nitrocellulose membrane. The next day the filters were washed gently under deionized H20, then denatured in 1X Western denaturing buffer (625 mM Tris, 625 mM glycine, pH9.0,5 mM P- mercaptoethanol) at 65°C for 10 minutes. Filters were blocked for 30 minutes in TTBS (160 mM NaCI, 20 mM Tris pH7.4,0.1% Tween 20) and 5% non-fat dry milk. The filters were then exposed to an anti-Glu-Glu tag antibody conjugated to horseradish peroxidase (5 gel of antibody diluted into 10 ml TTBS + 5% non-fat dry milk) at room temperature for 1 hour. Filters were washed twice for 5 minutes in TTBS with no milk and rinsed briefly in water. The filters were screened using commercially available chemiluminescence reagents (ECLTM direct labelling kit; Amersham Corp., Arlington Heights, IL) as a 1: 1 dilution, and the filters were immediately exposed to film. One clone produced a detectable signal.

Example 4 To generate a P. methanolica strain deficient for vacuolar proteases, the PEP4 and PRBI genes were identified and disrupted. PEP4 and PRBl sequences were amplified by PCR in reaction mixtures containing 100 pmol of primer DNA, 1X buffer as supplied (Boehringer Mannheim, Indianapolis, IN), 250 RM dNTPs, 1-100 pmol of

template DNA, and 1 unit of Taq polymerase in a reaction volume of 100 fil. The DNA was amplified over 30 cycles of 94°C, 30 seconds; 50°C, 60 seconds; and 72°C, 60 seconds.

Using an alignment of PEP4 sequences derived from S. cerevisiae (Ammerer et al., Mol. Cell. Biol. 6: 2490-2499,1986; Woolford et al., Mol. Cell. Biol.

6: 2500-2510,1986) and P. pastoris (Gleeson et al., U. S. Patent No. 5,324,660), several sense and antisense primers corresponding to conserved regions were designed. One primer set, ZC9118 (SEQ ID NO: 11) and ZC9464 (SEQ ID NO: 12) produced a PCR product of the expected size from genomic DNA, and this set was used to identify a genomic clone corresponding to the amplified region. DNA sequencing of a portion of this genomic clone (shown in SEQ ID NO: 13) revealed an open reading frame encoding a polypeptide (SEQ ID NO: 14) with 70% amino acid identity with proteinase A from S. cerevisiae.

Primers for the identification of P. methanolica PRB 1 were designed on the basis of alignments between the PRBI genes of S. cerevisiae (Moehle et al., Mol.

Cell. Biol. 7: 4390-4399,1987), P. pastoris (Gleeson et al., U. S. Pat. No. 5,324,660), and Kluyveromyces lactis (Fleer et al., WIPO Publication WO 94/00579). One primer set, ZC9126 (SEQ ID NO: 15) and ZC9741 (SEQ ID NO: 16) amplified a ca. 400 bp fragment from genomic DNA (SEQ ID NO: 17). This product was sequenced and found to encode a polypeptide (SEQ ID NO: 18) with 70% amino acid identity with proteinase B from S. cerevisiae. The PRB primer set was then used to identify a genomic clone encompassing the P. methanolica PRBI gene.

Deletion mutations in the P. methanolica PEP4 and PRBI genes were generated using available restriction enzyme sites. The cloned genes were restriction mapped. The pep4A allele was created by deleting a region of approximately 500 bp between BamHI and NcoI sites and including nucleotides 1 through 393 the sequence shown in SEQ ID NO: 13. The prblA allele was generated by deleting a region of approximately 1 kbp between NcoI and EcoRV sites and including the sequence shown in SEQ ID NO: 17. The cloned PEP4 and PRBI genes were subcloned into pCZR139, a phagemid vector (pBluescript0 II KS (+), Stratagene, La Jolla, CA) that carried a 2.4 kb SpeI ADE2 insert, to create the deletions. In the case of PEP4 gene, the unique BamHI site in pCZR139 was eliminated by digestion, fill-in, and religation. The vector was then linearized by digestion with EcoRI and Hindi, and a ca. 4 kb EcoRI-HindE fragment spanning the PEP4 gene was ligated to the linearized vector to produce plasmid pCZR142. A ca. 500 bp deletion was then produced by digesting pCZR142 with BamHI and NcoI, filling in the ends, and religating the DNA to produce plasmid pCZR143. The PRBI gene (-5 kb XhoI-BamHI fragment) was subcloned into

pCZR139, and an internal EcoRV-NcoI fragment, comprising the sequence shown in SEQ ID NO: 17, was deleted to produce plasmid pCZR153.

Plasmid pCZR143 was linearized with Asp718, which cut at a unique site. The linearized plasmid was introduced into the P. methanolica PMAD11 strain (an ade2 mutant generated as disclosed in U. S. Patent No. 5,736,383). Transformants were grown on-ADE DS (Table 1) to identify Ade+ transformants. Two classes of white, Ade+ transformants were analyzed. One class arose immediately on the primary transformation plate; the second became evident as rapidly growing white papillae on the edges of unstable, pink transformant colonies.

Southern blotting was used to identify transformants that had undergone the desired homologous integration event. 100 ul of cell paste was scraped from a 24- 48 hour YEPD plate and washed in 1 ml water. Washed cells were resuspended in 400 , of spheroplast buffer (1.2 M sorbitol, 10 mM Na citrate pH 7.5,10 mM EDTA, 10 mM DTT, 1 mg/ml zymolyase 100T) and incubated at 37°C for 10 minutes. Four hundred gel of 1 % SDS was added, the cell suspension was mixed at room temperature until clear, 300 u. l of 5 M potassium acetate was mixed in, and the mixture was clarified by microcentrifugation for 5 minutes. 750 uJ of the clarified lysate was extracted with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1), 600 fol was transferred to a fresh tube, 2 volumes of 100% ethanol was added, and the DNA was precipitated by microcentrifugation for 15 minutes at 4°C. The pellet was resuspended in 50 Rl of TE (10 mM Tris pH 8.0,1 mM EDTA) containing 100 KLg/ml of RNAase A.

Ten ul of DNA (approximately 100 ng) was digested in 100 gel total volume with appropriate enzymes, precipitated with 200 ul ethanol, and resuspended in 10 ul of DNA loading dye. The DNA was separated in 0.7% agarose gels and transferred to nylon membranes (Nytran N+, Amersham Corp., Arlington Heights, IL) in a semi-dry blotting apparatus (BioRad Laboratories, Richmond, CA) as recommended by the manufacturer. Transferred DNA was denatured, neutralized, and cross-linked to the membrane with UV light using a Stratalinker (Stratagene, La Jolla, CA). To identify strains with a tandem integration at PEP4, two probes were used. One was a 1400 bp EcoRI-HindE fragment from the 3'end of PEP4. The second was a 2000 bp BamHI -EcoRI fragment from the 5'end of PEP4. Fragments were detected using chemiluminescence reagents (ECL direct labelling kit; Amersham Corp., Arlington Heights, IL).

Parent strains harboring a tandem duplication of the wild-type and deletion alleles of the gene were grown in YEPD broth overnight to allow for the generation of looped-out, Ade-strains. These cells were then plated at a density of 2000-5000 colonies per plate on adenine-limited YEPD plates, grown for 3 days at

30°C and 3 days at room temperature. The shift to room temperature enhanced pigmentation of rare, pink, Ade-colonies. Loop-out strains were consistently detected at a frequency of approximately one pink, Ad'colon per 10,000 colonies screened.

These strains were screened for retention of the wild-type or mutant genes by Southern blotting or by PCR using primers that spanned the site of the deletion. An axe2-11 pep4A strain was designated PMAD15.

The PRBl gene was then deleted from PMAD15 essentially as described above by transformation with plasmid pCZR153. Blots were probed with PCR- generated probes for internal portions of the PRBI and ADE2 genes. The PRBI probe was generated by subcloning a 2.6 kb Clan-sel fragment of PRBI into the phagemid vector pBluescript II KS (+) (+) produce pCZR150, pCZR150, amplifying amplifying desired desired by PCR using primers ZC447 (SEQ ID NO: 19) and ZC976 (SEQ ID NO: 20). The ADE2 probe was generated by amplifying the ADE2 gene in pCZR139 with primers ZC9079 (SEQ ID NO: 21) and ZC9080 (SEQ ID NO: 22). The resulting ade2-ll pep4A prbl strain was designated PMAD 16.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.