AU718510B2 - Transformation of pichia methanolica - Google Patents

Description

WO 98/02565 PCT/US97/12581 1 Description TRANSFORMATION OF PICHIA METHANOLICA 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. Some methylotrophic yeasts have been shown to grow rapidly to high biomass on minimal defined media. Certain genes of methylotrophic yeasts are tightly regulated and highly expressed under induced or de-repressed conditions, suggesting that promoters of these genes might be useful for producing polypeptides of commercial value. See, for example, Faber et al., Yeast 11:1331, 1995; Romanos et al., Yeast 8:423, 1992; and Cregg et al., Bio/Technology 11:905, 1993.

Development of methylotrophic yeasts as hosts for use in recombinant production systems has been slow, due in part to a lack of suitable materials promoters, selectable markers, and mutant host cells) and methods transformation techniques). The most highly developed methylotrophic host systems utilize Pichia pastoris and Hansenula polymorpha (Faber et al., Curr. Genet. 25:305-310, 1994; Cregg et al., ibid.; Romanos et al., ibid.; U.S. Patent No. 4,855,242; U.S. Patent No. 4,857,467; U.S. Patent No. 4,879,231; and U.S. Patent No.

4,929,555).

There remains a need in the art for methods of transforming additional species of methylotrophic yeasts and for using the transformed cells to produce polypeptides of economic importance, including industrial enzymes and pharmaceutical proteins. The present invention provides such methods as well as other, related advantages.

Summary of the Invention The present invention provides methods for introducing DNA molecules into Pichia methanolica cells and cells transformed according to these methods.

Within one aspect of the invention, there is provided a method for introducing a DNA molecule into a Pichia methanolica cell, comprising exposing a Pichia methanolica cell, in the presence of a linear DNA molecule, to an exponentially decaying, pulsed electric field having a field strength of from to 4.5kV/cm and a time constant of from 15 to 40 milliseconds, whereby said DNA molecule is introduced into said cell. Within one embodiment of the invention, the DNA molecule comprises a segment encoding a polypeptide, other than a P. methanolica polypeptide, operably linked to a P.

methanolica gene transcription promoter and a P. methanolica gene transcription terminator. Within a preferred embodiment, the transcription promoter is a P. methanolica AUG1 gene promoter, which, within one embodiment, comprises a sequence of nucleotides as shown in SEQ ID NO:2 from nucleotide 24 to nucleotide 1354. The DNA molecule may further comprise a selectable marker gene that complements a mutation in the host cell. Within one embodiment, the selectable marker gene is a P. methanolica gene, such as a P. methanolica ADE2 gene.

Within a second aspect of the invention there are provided methods for transforming

P.

methanolica with heterologous DNA, comprising exposing a population of P. methanolica cells, in the presence of heterologous linear DNA molecules, to an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5kV/cm and a time constant of from 15 to 40 milliseconds, whereby the heterologous DNA is introduced into at least a portion of the population, and recovering cells into which the DNA has been introduced. Within one embodiment of the invention, from 103 to 105 cells are recovered per microgram of heterologous DNA. Within another embodiment, from 0.9 x 20 104 to 1.1 x 104 cells are recovered per microgram of heterologous DNA. Within a further embodiment, the methods further comprise the step of recovering integrative transformants from the recovered cells, such as by culturing the cells in a growth medium comprising sorbitol as a carbon source. Within an additional embodiment, the population of cells is in early log phase growth.

0: Within other aspects, the present invention provides Pichia methanolica cells transformed by 25 the methods disclosed above.

These and other aspects of the invention will become evident upon reference to the following S0 detailed description and the attached drawings.

Brief Description of the Drawings Fig. 1 illustrates the effects of field strength and pulse duration on electroporation efficiency of P. methanolica.

Fig. 2 is a schematic diagram of a recombination event between plasmid-pCZR140 and P.

methanolica genomic DNA.

Fig. 3 is a schematic diagram of a recombination event between plasmid pCZR137 and P.

methanolica genomic DNA.

Detailed Description of the Invention WO 98/02565 PCT/US97/12581 3 Prior to setting forth the invention in more detail, it will be useful to define certain terms used herein: Early log phase growth--That phase of cellular growth in culture when the cell concentration is from 2 x 10 6 cells/ml to 8 x 10 6 cells/ml.

Heterologous DNA--A DNA molecule, or a population of DNA molecules, that does not exist naturally within a given host cell. DNA molecules heterologous to a particular host cell may contain DNA derived from the host cell species so long as that host DNA is combined with non-host DNA. For example, a DNA molecule containing a non-host DNA segment encoding a polypeptide operably linked to a host DNA segment comprising a transcription promoter is considered to be a heterologous DNA molecule.

Integrative transformants--Cells into which have been introduced heterologous DNA, wherein the heterologous DNA has become integrated into the genomic DNA of the cells.

Linear DNA--DNA molecules having free 5' and 3' ends, that is non-circular DNA molecules. Linear DNA can be prepared from closed circular DNA molecules, such as plasmids, by enzymatic digestion or physical disruption.

Operably linked--The term operably linked indicates that DNA segments are arranged so that they function in concert for their intended purposes, transcription initiates in the promoter and proceeds through the coding segment to the terminator.

As noted above, the present invention provides methods for introducing DNA into cells of the methylotrophic yeast Pichia methanolica, and for selecting cells into which the DNA has been introduced. Those skilled in the art will recognize that transformation of cells with both homologous DNA (DNA from the host species) and heterologous DNA is a prerequisite to a large number of diverse biological applications. The methods of the present invention are particularly well suited to the preparation of cells transformed with heterologous DNA, which cells can be used for the production of polypeptides and proteins, including polypeptides and proteins of higher organisms, including humans. The present invention further provides for the transformation of Pichia methanolica cells with other DNA molecules, including DNA libraries and synthetic DNA molecules. The invention thus provides techniques that can be used to express genetically diverse libraries to produce products that are screened for novel biological activities, to engineer cells for use as targets for the screening of compound libraries, and to genetically modify cells to enhance their utility within other processes.

Strains of Pichia methanolica are available from the American Type Culture Collection (Rockville, MD) and other repositories. Within one embodiment of the invention, cells to be transformed with heterologous DNA will have a mutation that can be complemented by a gene (a "selectable marker") on the heterologous DNA molecule. This selectable marker allows the transformed cells to grow under conditions in which untransformed cells cannot multiply ("selective conditions"). The general principles of selection are well known in the art.

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 WO 98/02565 PCTIUS97/12581 4 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 Pichia methanolica is a P. methanolica ADE2 gene, which encodes aminoimidazole carboxylase (AIRC; EC 4.1.1.21). 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: 1. The sequence illustrated includes 1006 nucleotides of 5' non-coding sequence and 442 nucleotides of 3' noncoding 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. Those skilled in the art will recognize that this and other sequences provided herein represent single alleles of the respective genes, and that allelic variation is expected to exist. Any functional ADE2 allele can be used within the present invention. Other nutritional markers that can be used within the present invention include the P. methanolica ADE1, HIS3, and LEU2 genes, which allow for selection in the absence of adenine, histidine, and leucine, respectively. For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (AUG1 and AUG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes (PEP4 and PRB1) are preferred. Genedeficient mutants can be prepared by known methods, such as site-directed mutagenesis. P.

methanolica genes can be cloned on the basis of homology with their counterpart Saccharomyces cerevisiae genes. The ADE2 gene disclosed herein was given its designation on the basis of such homology.

Within another embodiment of the invention, a dominant selectable marker is used, thereby obviating the need for mutant host cells. Dominant selectable markers are those that are able to provide a growth advantage to wild-type cells. Typical dominant selectable markers are genes that provide resistance to antibiotics, such as neomycin-type antibiotics G418), hygromycin B, and bleomycin/phleomycin-type antibiotics 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 Electroporation is used within the present invention to facilitate the introduction of DNA into P. methanolica cells. Electroporation is the process of using a pulsed electric field to transiently permeabilize cell membranes, allowing macromolecules, such as DNA, to pass into cells. Electroporation has been described for use with mammalian Neumann et al., EMBO J. 1:841-845, 1982) and fungal Meilhoc et al., Bio/Technology 8:223-227, 1990) host cells.

However, the actual mechanism by which DNA is transferred into the cells is not well understood. For transformation of P. methanolica, it has been found that electroporation is surprisingly efficient when the cells are exposed to an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5kV/cm and a time constant of from to 40 milliseconds. The time constant r is defined as the time required for the initial peak voltage V o to drop to a value of Vo/e. The time constant can be calculated as the product of the total resistance and capacitance of the pulse circuit, T R x C. Typically, resistance and capacitance are either preset or may be selected by the user, depending on the electroporation equipment selected. In any event, the equipment is configured in accordance with the manufacturer's instructions to provide field strength and decay parameters as disclosed above. Electroporation equipment is available from commercial suppliers BioRad Laboratories, Hercules, CA).

DNA molecules for use in transforming P. methanoclica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation. For polypeptide or protein production, the DNA molecules will include, in addition to the selectable marker disclosed above, an expression casette comprising a transcription promoter, a DNA segment a cDNA) encoding the polypeptide or protein of interest, and a transcription terminator. These elements are operably linked to provide for transcription of the DNA segment of interest. It is preferred that the promoter and terminator be that of a P. methanoclica gene. A preferred promoter is that of a P. methanoclica alcohol utilization gene (AUG1), a representative coding strand sequence of which is shown in SEQ ID NO:2. Within SEQ ID NO:2, the initiation ATG °*o6 .20 codon is at nucleotides 1355-1357. Nucleotides 1-23 of SEQ ID NO:2 are non-AUG] polylinker sequence. It is particularly preferred to utilize as a promoter a segment comprising nucleotides 24-1354 of SEQ ID NO:2, although additional upstream sequence can be included. P. methanolica contains a second alcohol utilization gene, AUG2, the promoter of which can be used within the present invention. Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. The DNA molecules will further include a selectable marker to allow for identification, selection, and maintenance of transformants. The DNA molecules may further contain additional elements, such an origin of replication and a selectable marker that allow amplification and maintenance of the DNA in an alternate host E. coli). To facilitate integration of the DNA into the host chromosome, it is preferred to have the entire expression segment, comprising the promoter--gene of interest--terminator plus selectable marker, flanked at both ends by host DNA sequences.

This is conveniently accomplished by including 3' untranslated DNA sequence at the downstream end of the expression segment and relying on the promoter sequence at the 35 end. When using linear DNA, the expression segment will be flanked by cleavage sites to allow for linearization of the molecule and separation of the expression segment form other sequences a bacterial origin of replication and selectable marker). Preferred such cleavage sites are those that are recognized by restriction endonucleases that cut infrequently within a DNA sequence, such as those that recognize 8-base target sequences e Not I).

[n:/libxx]01101:bmv WO 98/02565 PCTIUS97/12581 6 Proteins that can be produced in P. methanolica using the methods of the present invention include proteins of industrial and 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, fibroblast growth factors, and epidermal growth factor; cytokines such as erythropoietin and thrombopoietin; and hormones such as insulin, leptin, and glucagon.

For use within the present invention, P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25 0 C to 35 0 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 is YEPD (Table The cells may be passaged by dilution into fresh culture medium or stored for short periods on plates under refrigeration. For long-term storage, the cells are preferably kept in a 50% glycerol solution at -70 0

C.

Table 1

YEPD

2% D-glucose 2% BactoTM Peptone (Difco Laboratories, Detroit, MI) 1% BactoTM yeast extract (Difco Laboratories) 0.004% adenine 0.006% L-leucine ADE D 0.056% -Ade -Trp -Thr powder 0.67% yeast nitrogen base without amino acids 2% D-glucose 200X tryptophan, threonine solution ADE DS 0.056% -Ade -Trp -Thr powder 0.67% yeast nitrogen base without amino acids 2% D-glucose 200X tryptophan, threonine solution 18.22% D-sorbitol LEU D 0.052% -Leu -Trp -Thr powder 0.67% yeast nitrogen base without amino acids 2% D-glucose 200X tryptophan, threonine solution HIS D 0.052% -His -Trp -Thr powder 0.67% yeast nitrogen base without amino acids WO 98/02565 7 2% D-glucose 200X tryptophan, threonine solution URA D 0.056% -Ura -Trp -Thr powder 0.67% yeast nitrogen base without amino acids 2% D-glucose 200X tryptophan, threonine solution PCTIUS97/12581 WO 98/02565 PCTIUS97/12581 8 Table 1, continued URA DS 0.056% -Ura -Trp -Thr powder 0.67% yeast nitrogen base without amino acids 2% D-glucose 200X tryptophan, threonine solution 18.22% D-sorbitol -Leu -Trp -Thr powder powder made by combining 4.0 g adenine, 3.0 g arginine, 5.0 g aspartic acid, 2.0 g histidine, 6.0 g isoleucine, 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) -His -Trp -Thr powder powder made by combining 4.0 g adenine, 3.0 g arginine, 5.0 g aspartic acid, 6.0 g isoleucine, 8.0 g leucine, 4.0 g lysine, 2.0 g methionine, 6.0 g phenylalanine, 5.0 g serine, g tyrosine, 4.0 g uracil, and 6.0 g valine (all L- amino acids) -Ura -Trp -Thr powder powder made by combining 4.0 g adenine, 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, and 6.0 g valine (all L- amino acids) -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, g tyrosine, 4.0 g uracil, and 6.0 g valine (all L- amino acids) 200X trvptophan, threonine solution L-threonine, 0.8% L-tryptophan in H 2 0 For plates, add 1.8% BactoTM agar (Difco Laboratories) Electroporation of P. methanolica is preferably carried out on cells in early log phase growth. Cells are streaked to single colonies on solid media, preferably solid YEPD.

After about 2 days of growth at 30 0 C, single colonies from a fresh plate are used to inoculate the desired volume of rich culture media YEPD) to a cell density of about 5 10 x 105 cells/ml. Cells are incubated at about 25 35 0 C, preferably 30 0 C, with vigorous shaking, until they are in early log phase. The cells are then harvested, such as by centrifugation at 3000 x g for 2-3 minutes, and resuspended. Cells are made electrocompetent by reducing disulfide bonds in the cell walls, equilibrating them in an ionic solution that is compatible with the electroporation conditions, and chilling them. Cells are typically made electrocompetent by incubating them in a buffered solution at pH 6-8 containing a reducing agent, such as dithiothreitol (DTT) or 13-mercaptoethanol (BME), to reduce cell wall proteins to facilitate subsequent uptake of DNA. A preferred incubation buffer in this regard is a fresh solution of mM potassium phosphate buffer, pH 7.5, containing 25 mM DTT. The cells are incubated in this buffer (typically using one-fifth the original culture volume) at about 30 0 C for about 5 to minutes, preferably about 15 minutes. The cells are then harvested and washed in a suitable electroporation buffer, which is used ice-cold. Suitable buffers in this regard include pH 6-8 solutions containing a weak buffer, divalent cations Mg Ca and an osmotic stabilizer a sugar). After washing, the cells are resuspended in a small volume of the buffer, at which time they are electrocompetent and can be used directly or aliquotted and stored frozen (preferably at -70 0 A preferred electroporation buffer is STM (270 mM sucrose, 10 mM Tris, pH 7.5, 1 mM MgCl 2 Within a preferred protocol, the cells are subjected to two washes, first in the original culture volume of ice-cold buffer, then in one-half the original volume. Following the second wash, the cells are harvested and resuspended, typically using about 3-5 ml of buffer for an original culture volume of 200 ml.

Electroporation is carried out using a small volume of electrocompetent cells (typically about 100 plA) and up to one-tenth volume of linear DNA molecules. For example, 0.1 ml of cell suspension in a buffer not exceeding 50 mM in ionic strength is combined with 0.1-10 [pg of DNA (vol. 5 10 tl). This mixture is placed in an ice-cold electroporation cuvette and subjected to a pulsed electric field of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a tme constant of from 15 to 40 milliseconds, preferably 15-30 milliseconds, more preferably 15-25 illiseconds, most preferably about 20 milliseconds, with exponential decay. The actual equipment settings used to achieve the desired pulse parameters will be determined by the .equipment used. When using a BioRad (Hercules, CA) Gene PulserTM electroporator with a 2 :*mm electroporation cuvette, resistance is set at 600 ohms or greater, preferably "infinite" .resistance, and capacitance is set at 25 pF to obtain the desired field characteristics. After being pulsed, the cells are diluted approximately 10X into 1 ml of YEPD broth and incubated at 30 0

C

for one hour.

The cells are then harvested and plated on selective media. Within a preferred Smbodiment, the cells are washed once with a small volume (equal to the diluted volume of the 4lectroporated cells) of IX yeast nitrogen base (6.7 g/L yeast nitrogen base without amino acids; Difco Laboratories, Detroit, MI), and plated on minimal selective media. Cells having an ade2 ornutation that have been transformed with an ADE2 selectable marker can be plated on a minimal medium that lacks adenine, such as ADE D (Table 1) or ADE DS (Table In a typical procedure, 250 gl aliqouts of cells are plated on 4 separate ADE D or ADE DS plates to select *for Ade cells.

O P. methanolica recognizes certain infrequently occuring sequences, termed autonomously replicating sequences (ARS), as origins of DNA replication, and these sequences may fortuitously occur within a DNA molecule used for transformation, allowing the transforming DNA to be maintained extrachromosomally. However, integrative transformants are generally preferred for use in protein production systems. Integrative transformants have a P RA found growth advantage over ARS transformants on selective media containing sorbitol as a -o ^wV o^ WO 98/02565 PCT/US97/12581 carbon source, thereby providing a method for selecting integrative transformants from among a population of transformed cells. ARS sequences have been found to exist in the ADE2 gene and, possibly, the A UG1 gene of P. methanolica. ade2 host cells of Pichia methanolica transformed with an ADE2 gene can thus become Ade+ by at least two different modes. The ARS within the ADE2 gene allows unstable extrachromosomal maintenance of the transforming DNA (Hiep et al., Yeast 9:1189-1197, 1993). Colonies of such transformants are characterized by slower growth rates and pink color due to prolific generation of progeny that are Ade-. Transforming DNA can also integrate into the host genome, giving rise to stable transformants that grow rapidly, are white, and that fail to give rise to detectable numbers of Ade- progeny. ADE D plates allow the most rapid growth of transformed cells, and unstable and stable transformants grow at roughly the same rates. After 3-5 days of incubation on ADE D plates at 30 0 C stable transformant colonies are white and roughly twice the size of unstable, pink transformants. ADE DS plates are more selective for stable transformants, which form large mm) colonies in 5-7 days, while unstable (ARS-maintained) colonies are much smaller (1l mm). The more selective ADE DS media is therefore preferred for the identification and selection of stable transformants.

For some applications, such as the screening of genetically diverse libraries for rare combinations of genetic elements, it is sometimes desirable to screen large numbers of unstable transformants, which have been observed to outnumber stable transformants by a factor of roughly 100. In such cases, those skilled in the art will recognize the utility of plating transformant cells on less selective media, such as ADE D.

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 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 and differences in promoter activity among individual isolates. It is therefore advantageous to screen a number of isolates for expression level prior to selecting a production strain. A variety of suitable screening methods are available. For example, transformant colonies are grown on plates that are overlayed with membranes 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 WO 98/02565 PCT/US97/12581 11 will be determined in part by the protein of interest. Such methods are readily selected and practiced by the skilled practitioner.

For small-scale protein production plate or shake flask production), P.

methanolica transformants that carry an expression cassette comprising a methanol-regulated promoter (such as the A UG1 promoter) are grown in the presence of methanol and the absence of interfering amounts of other carbon sources glucose). For small-scale experiments, including preliminary screening of expression levels, transformants may be grown at 30°C on solid media containing, for example, 20 g/L Bacto-agar (Difco), 6.7 g/L yeast nitrogen base without amino acids (Difco), 10 g/L methanol, 0.4 tg/L biotin, and 0.56 g/L of -Ade -Thr -Trp powder. Because methanol is a volatile carbon source it is readily lost on prolonged incubation.

A continuous supply of methanol can be provided by placing a solution of 50% methanol in water in the lids of inverted plates, whereby the methanol is transferred to the growing cells by evaporative transfer. In general, not more than 1 mL of methanol is used per 100-mm plate.

Slightly larger scale experiments can be carried out using cultures grown in shake flasks. In a typical procedure, cells are cultivated for two days on minimal methanol plates as disclosed above at 30 0 C, then colonies are used to inoculate a small volume of minimal methanol media (6.7 g/L yeast nitrogen base without amino acids, 10 g/L methanol, 0.4 gpg/L biotin) at a cell density of about 1 x 106 cells/ml. Cells are grown at 30 0 C. Cells growing on methanol have a high oxygen requirement, necessitating vigorous shaking during cultivation. Methanol is replenished daily (typically 1/100 volume of 50% methanol per day).

For production scale culturing, fresh cultures of high producer clones are prepared in shake flasks. The resulting cultures are then used to inoculate culture medium in a fermenter.

Typically, a 500 ml culture in YEPD grown at 30 0 C for 1-2 days with vigorous agititation is used to inoculate a 5-liter fermenter. The cells are grown in a suitable medium containing salts, glucose, biotin, and trace elements at 28 0 C, pH 5.0, and >30% dissolved 02. After the initial charge of glucose is consumed (as indicated by a decrease in oxygen consumption), a glucose/methanol feed is delivered into the vessel to induce production of the protein of interest.

Because large-scale fermentation is carried out under conditions of limiting carbon, the presence of glucose in the feed does not repress the methanol-inducible promoter.

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

Examples Example 1 P. methanolica cells (strain CBS6515 from American Type Culture Collection, Rockville, MD) were mutagenized by UV exposure. A killing curve was first generated by plating cells onto several plates at approximately 200-250 cells/plate. The plates were then exposed to UV radiation using a G8T5 germicidal lamp (Sylvania) suspended 25 cm from the surfaces of the plates for periods of time as shown in Table 2. The plates were then protected from visible light sources and incubated at 30 0 C for two days.

WO 98/02565 PCT/IJS97/12581 12 Table 2 Viable Cells Time Plate 1 Plate 2 Average 0 sec. 225 229 227 1 sec. 200 247 223 2 sec. 176 185 181 4 sec. 149 86 118 8 sec. 20 7 14 16 sec. 0 2 1 Large-scale mutagenesis was then carried out using a 2-second UV exposure to provide about 20% killing. Cells were plated at approximately 104 cells/plate onto eight YEPD plates that were supplemented with 100 mg/L each of uracil, adenine, and leucine, which were added to supplement the growth of potential auxotrophs having the cognate deficiencies.

Following UV exposure the plates were wrapped in foil and incubated overnight at 30 0 C. The following day the colonies on the plates (~105 total) were resuspended in water and washed once with water. An amount of cell suspension sufficient to give an OD 6 0 0 of 0.1 0.2 was used to inoculate 500 ml of minimal broth made with yeast nitrogen base without amino acids or ammonia, supplemented with 1% glucose and 400 tg/L biotin. The culture was placed in a 2.8 L baffled Bell flask and shaken vigorously overnight at 30°C. The following day the cells had reached an OD 6 0 0 of -1.0 2.0. The cells were pelleted and resuspended in 500 ml of minimal broth supplemented with 5 g/L ammonium sulfate. The cell suspension was placed in a 2.8 L baffled Bell flask and shaken vigorously at 30 0 C for 6 hours. 50 ml of the culture was set aside in a 250-ml flask as a control, and to the remainder of the culture was added 1 mg nystatin to select for auxotrophic mutants (Snow, Nature 211:206-207, 1966). The cultures were incubated with shaking for an additional hour. The control and nystatin-treated cells were then harvested by centrifugation and washed with water three times. The washed cells were resuspended to an

OD

6 0 0 of 1.0 in 50% glycerol and frozen. Titering of nystatin-treated cells versus the control cells for colony forming units revealed that nystatin enrichment had decreased the number of viable cells by a factor of 104.

10-2 dilutions of nystatin-treated cells were plated on 15 YEPD plates. Colonies were replica-plated onto minimal plates agar, 1 x YNB, 2% glucose, 400 gg/L biotin). The frequency of auxotrophs was about 2 Approximately 180 auxotrophic colonies were picked to YEPD Ade, Leu, Ura plates and replica-plated to various dropout plates. All of the auxotrophs were Ade-. Of these, 30 were noticably pink on dropout plates (LEU D, HIS D, etc.; see Table Of the 30 pink mutants, 21 were chosen for further study; the remainder were either leaky for growth on ADE D plates or contaminated with wild-type cells.

WO 98/02565 PCT/US97/12581 The Ade- mutants were then subjected to complementation analysis and phenotypic testing. To determine the number of loci defined by the mutants, all 21 mutants were mated to a single pink, Ade- tester strain (strain Mating was carried out by mixing cell suspensions (OD 6 0 0 1) and plating the mixtures in 10 tl aliquots on YEPD plates. The cells were then replicated to SPOR media Na acetate, 1% KC1, 1% glucose, 1% agar) and incubated overnight at 30 0 C. The cells were then replica-plated to ADE D plates for scoring of phenotype. As shown in Table 3, some combinations of mutants failed to give Ade+ colonies (possibly defining the same genetic locus as in strain while others gave rise to numerous Ade+ colonies (possibly defining a separate genetic locus). Because mutant #3 gave Ade+ colonies when mated to complementation testing was repeated with mutant If the group of mutants defined two genetic loci, then all mutants that failed to give Ade+ colonies when mated to strain #2 should give Ade+ colonies when mated to Results of the crosses are shown in Table 3.

Table 3 Mutant x Mutant #2 x Mutant #3 #1 #3 #18 #24 #28 #2 #6 #8 #9 #11 #17 #19 #22 #27 #4 #12 #16 WO 98/02565 PCT/US97/12581 14 As shown in Table 3, most mutants fell into one of two groups, consistent with the idea that there are two adenine biosynthetic genes that, when missing, result in pink colonies on limiting adenine media. Three colonies #12, and #16) may either define a third locus or exhibit intragenic complementation. Two intensely pigmented mutants from each of the two complementation groups and #10; #6 and #11) were selected for further characterization.

Additional analysis indicated that Ade- was the only auxotrophy present in these strains.

A P. methanolica clone bank was constructed in the vector pRS426, a shuttle vector comprising 2ut 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 sizefractionated 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 in E. coli MC 1061 cells using a BioRad Gene PulserTM device as recommended by the manufacturer.

The genomic library was used to transform S. cerevisiae strain HBY21A (ade2 ura3) by electroporation (Becker and Guarente, Methods Enzymol. 194:182-187, 1991). The cells were resuspended in 1.2 M sorbitol, and six 300-tl aliquots were plated onto ADE D, ADE DS, URA D and URA DS plates (Table Plates were incubated at 30 0 C for 4-5 days. No Ade colonies were recovered on the ADE D or ADE DS plates. Colonies from the URA D and URA DS plates were replica-plated to ADE D plates, and two closely spaced, white colonies were obtained. These colonies were restreaked and confirmed to be Ura+ and Ade These two strains, designated Adel and Ade6, were streaked onto media containing 5 FOA (5 fluoro orotic acid; Sikorski and Boeke, Methods Enzymol. 194:302-318). Ura- colonies were obtained, which were found to be Ade- upon replica plating. These results indicate that the Ade+ complementing activity is genetically linked to the plasmid-borne URA3 marker. Plasmids obtained from yeast strains Adel and Ade6 appeared to be identical by restriction mapping as described below.

These genomic clones were designated pADE -1 and pADE1-6, respectively.

Total DNA was isolated from the HBY21A transformants Adel and Ade6 and used to transform E. coli strain MC 1061 to AmpR. DNA was prepared from 2 Amp R colonies of Adel and 3 AmpR colonies of Ade6. The DNA was digested with Pst I, Sca I, and Pst I Sca I and analyzed by gel electrophoresis. All five isolates produced the same restriction pattern.

PCR primers were designed from the published sequence of the P. methanolica ADE2 gene (also known as ADE1; Hiep et al., Yeast 9:1251-1258, 1993). Primer 9080 (SEQ ID WO 98/02565 PCT/US97/12581 NO:3) was designed to prime at bases 406-429 of the ADE2 DNA (SEQ ID NO:1), and primer 9079 (SE ID NO:4) was designed to prime at bases 2852-2829. Both primers included tails to introduce Avr II and Spe I sites at each end of the amplified sequence. The predicted size of the resulting PCR fragment was 2450 bp.

PCR was carried out using plasmid DNA from the five putative ADE2 clones as template DNA. The 100 [il reaction mixtures contained lx Taq PCR buffer (Boehringer Mannheim, Indianapolis, IN), 10-100 ng of plasmid DNA, 0.25 mM dNTPs, 100 pmol of each primer, and 1 4l Taq polymerase (Boehringer Mannheim). PCR was run for 30 cycles of seconds at 94 0 C, 60 seconds at 50 0 C, and 120 seconds at 72 0 C. Each of the five putative ADE2 genomic clones yielded a PCR product of the expected size (2.4 kb). Restriction mapping of the DNA fragment from one reaction gave the expected size fragments when digested with Bgl II or Sal I.

The positive PCR reactions were pooled and digested with Spe I. Vector pRS426 was digested with Spe I and treated with calf intestinal phosphatase. Four gl of PCR fragment and 1 gl of vector DNA were combined in a 10 pl reaction mix using conventional ligation conditions. The ligated DNA was analyzed by gel electrophoresis. Spe I digests were analyzed to identify plasmids carrying a subclone of the ADE2 gene within pRS426. The correct plasmid was designated pCZR1 18.

Because the ADE2 gene in pCZRl 18 had been amplified by PCR, it was possible that mutations that disabled the functional character of the gene could have been generated. To test for such mutations, subclones with the desired insert were transformed singly into Saccharomyces cerevisiae strain HBY21A. Cells were made electrocompetent and transformed according to standard procedures. Transformants were plated on URA D and ADE D plates.

Three phenotypic groups were identified. Clones 1, 2, 11, and 12 gave robust growth of many transformants on ADE D. The transformation frequency was comparable to the frequency of Ura- transformants. Clones 6, 8, 10, and 14 also gave a high efficiency of transformation to both Ura and Ade+, but the Ade colonies were somewhat smaller than those in the first group.

Clone 3 gave many Ura colonies, but no Ade+ colonies, suggesting it carried a non-functional ade2 mutation. Clones 1, 2, 11, and 12 were pooled.

To identify the P. methanolica ade2 complementation group, two representative mutants from each complementation group and #10; #6 and which were selected on the basis of deep red pigmentation when grown on limiting adenine, were transformed with the cloned ADE gene. Two hundred ml cultures of early log phase cells were harvested by centrifugation at 3000 x g for 3 minutes and resuspended in 20 ml of fresh KD buffer (50 mM potassium phosphate buffer, pH 7.5, containing 25 mM DTT). The cells were incubated in this buffer at 30 0 C for 15 minutes. The cells were then harvested and resuspended in 200 ml of icecold STM (270 mM sucrose, 10 mM Tris, pH 7.5, 1 mM MgCl 2 The cells were harvested and resuspended in 100 ml of ice-cold STM. The cells were again harvested and resuspended in ml of ice-cold STM. 100- pl aliquouts of electrocompetent cells from each culture were then WO 98/02565 PCT[US97/12581 16 mixed with Not I-digested pADEI-1 DNA. The cell/DNA mixture was placed in a 2 mm electroporation cuvette and subjected to a pulsed electric field of 5 kV/cm using a BioRad Gene PulserTM set to 10002 resistance and capacitance of 25 pF. After being pulsed, the cells were diluted by addition of 1 ml YEPD and incubated at 30 0 C for one hour. The cells were then harvested by gentle centrifugation and resuspended in 400 pl minimal selective media lacking adenine (ADE The resuspended samples were split into 2 0 0-ul aliqouts and plated onto ADE D and ADE DS plates. Plates were incubated at 30 0 C for 4-5 days. Mutants #6 and #11 gave Ade+ transformants. No Ade transformants were observed when DNA was omitted, hence the two isolates appeared to define the ade2 complementation group. The ADE2 sequence is shown in SEQ ID NO:1.

Example 2 The P. methanolica clone bank disclosed in Example I was used as a source for cloning the Alcohol Utilization Gene (A UG1). The clone bank was stored as independent pools, each representing about 200-250 individual genomic clones. 0.1 il of "miniprep" DNA from each pool was used as a template in a polymerase chain reaction with PCR primers (8784, SEQ ID NO:5; 8787, SEQ ID NO:6) that were designed from an alignment of conserved sequences in alcohol oxidase genes from Hansenula polymorpha, Candida boidini, and Pichia pastoris. The amplification reaction was run for 30 cycles of 94°C, 30 seconds; 50 0 C, 30 seconds; 72 0 C, seconds; followed by a 7 minute incubation at 72 0 C. One pool gave a -600 bp band. DNA sequencing of this PCR product revealed that it encoded an amino acid sequence with sequence identity with the Pichia pastoris alcohol oxidase encoded by the AOX1 gene and about sequence identity with the Hansenula polymorpha alcohol oxidase encoded by the MOX1 gene. The sequence of the cloned A UG1 gene is shown in SEQ ID NO:2.

Sub-pools of pool #5 were analyzed by PCR using the same primers used in the initial amplification. One positive sub-pool was further broken down to identify a positive colony. This positive colony was streaked on plates, and DNA was prepared from individual colonies. Three colonies gave identical patterns after digestion with Cla I.

Restriction mapping of the genomic clone and PCR product revealed that the AUG1 gene lay on a 7.5 kb genomic insert and that sites within the PCR fragment could be uniquely identified within the genomic insert. Because the orientation of the gene within the PCR fragment was known, the latter information provided the approximate location and direction of transcription of the A UG1 gene within the genomic insert. DNA sequencing within this region revealed a gene with very high sequence similarity at the amino acid level to other known alcohol oxidase genes.

Example 3 ade2 mutant P. methanolica cells are transformed by electroporation essentially as disclosed above with an expression vector comprising the AUG1 promoter and terminator, WO 98/02565 PCT/US97/12581 17 human GAD65 DNA (Karlsen et al., Proc. Natl. Acad. Sci. USA 88:8337-8341, 1991), and ADE2 selectable marker. Colonies are patched to agar minimal methanol plates (10 to 100 colonies per 100-mm plate) containing 20 g/L BactoT'-agar (Difco), 6.7 g/L yeast nitrogen base without amino acids (Difco), 10 g/L methanol, and 0.4 pg/L biotin. The agar is overlayed with nitrocellulose, and the plates are inverted over lids containing 1 ml of 50% methanol in water and incubated for 3 to 5 days at 30 0 C. The membrane is then transferred to a filter soaked in 0.2 M NaOH, 0.1% SDS, 35 mM dithiothreitol to lyse the adhered cells. After 30 minutes, cell debris is rinsed from the filter with distilled water, and the filter is neutralized by rinsing it for minutes in 0.1 M acetic acid.

The filters are then assayed for adhered protein. Unoccupied binding sites are blocked by rinsing in TTBS-NFM (20 mM Tris pH 7.4, 0.1% Tween 20, 160 mM NaCl, powdered nonfat milk) for 30 minutes at room temperature. The filters are then transferred to a solution containing GAD6 monoclonal antibody (Chang and Gottlieb, J. Neurosci. 8:2123-2130, 1988), diluted 1:1000 in TTBS-NFM. The filters are incubated in the antibody solution with gentle agitation for at least one hour, then washed with TTBS (20 mM Tris pH 7.4, 0.1% Tween 160 mM NaCI) two times for five minutes each. The filters are then incubated in goat antimouse antibody conjugated to horseradish peroxidase (1 jtg/ml in TTBS-NFM) for at least one hour, then washed three times, 5 minutes per wash with TTBS. The filters are then exposed to commercially available chemiluminescence reagents (ECLTM; Amersham Inc., Arlington Heights, IL). Light generated from positive patches is detected on X-ray film.

To more accurately detect the level of GAD 6 5 expression, candidate clones are cultured in shake flask cultures. Colonies are grown for two days on minimal methanol plates at 0 C as disclosed above. The colonies are used to inoculate 20 ml of minimal methanol media (6.7 g/L yeast nitrogen base without amino acids, 10 g/L methanol, 0A tg/L biotin) at a cell density of 1 x 106 cells/ml. The cultures are grown for 1-2 days at 30 0 C with vigorous shaking.

0.2 ml of 50% methanol is added to each culture daily. Cells are harvested by centrifugation and suspended in ice-cold lysis buffer (20 mM Tris pH 8.0, 40 mM NaCl, 2 mM PMSF, 1 mM EDTA, 1 ptg/ml leupeptin, 1 jig/ml pepstatin, 1 jig/ml aprotinin) at 10 ml final volume per 1 g cell paste. 2.5 ml of the resulting suspension is added to 2.5 ml of 400-600 micron, ice-cold, acid-washed glass beads in a 15-ml vessel, and the mixture is vigorously agitated for one minute, then incubated on ice for 1 minute. The procedure is repeated until the cells have been agitated for a total of five minutes. Large debris and unbroken cells are removed by centrifugation at 1000 x g for 5 minutes. The clarified lysate is then decanted to a clean container. The cleared lysate is diluted in sample buffer SDS, 8 M urea, 100 mM Tris pH 6.8, 10% glycerol, 2 mM EDTA, 0.01% bromphenol glue) and electrophoresed on a 4-20% acrylamide gradient gel (Novex, San Diego, CA). Proteins are blotted to nitrocellose and detected with GAD6 antibody as disclosed above.

Clones exhibiting the highest levels of methanol-induced expression of foreign protein in shake flask culture are more extensively analyzed under high cell density fermentation WO 98/02565 PCT/US97/12581 18 conditions. Cells are first cultivated in 0.5 liter of YEPD broth at 30 0 C for 1 2 days with vigorous agitation, then used to inoculate a 5-liter fermentation apparatus BioFlow III; New Brunswick Scientific Co., Inc., Edison, NJ). The fermentation vessel is first charged with mineral salts by the addition of 57.8 g (NH 4 2

SO

4 68 g KH 2

PO

4 30.8 g MgSO 4 -7H 2 0, 8.6 g CaSO 4 -2H 2 0, 2.0 g NaCl, and 10 ml antifoam (PPG). H 2 0 is added to bring the volume to L, and the solution is autoclaved 40 minutes. After cooling, 350 ml of 50% glucose, 250 ml X trace elements (Table 25 ml of 200 pg/ml biotin, and 250 ml cell inoculum are added.

WO 98/02565 PCT/US97/12581 19 Table 4 X trace elements: FeSO 4 -7H 2 0 100mM 27.8 g/L CuSO 4 -5H 2 0 2mM 0.5 g/L ZnC1 2 8mM 1.09 g/L MnSO 4

-H

2 0 8mM 1.35 g/L CoC12-6H 2 0 2mM 0.48 g/L Na2MoO 4 .2H 2 0 ImM 0.24 g/L

H

3

BO

3 8mM 0.5 g/L KI 0.5mm 0.08 g/L biotin thiamine 0.5 g/L Add 1-2 mls H 2

SO

4 per liter to bring compounds into solution.

The fermentation vessel is set to run at 28 0 C, pH 5.0, and >30% dissolved 02.

The cells will consume the initial charge of glucose, as indicated by a sharp demand for oxygen during glucose consumption followed by a decrease in oxygen consumption after glucose is exhausted. After exhaustion of the initial glucose charge, a glucose-methanol feed supplemented with NH 4 and trace elements is delivered into the vessel at 0.2% glucose, 0.2% (w/v) methanol for 5 hours followed by 0.1% glucose, 0.4% methanol for 25 hours. A total of 550 grams of methanol is supplied through one port of the vessel as pure methanol using an initial delivery rate of 12.5 ml/hr and a final rate of 25 ml/hr. Glucose is supplied through a second port using a 700 ml solution containing 175 grams glucose, 250 ml 10X trace elements, and 99 g (NH 4 2

SO

4 Under these conditions the glucose and methanol are simultaneously utilized, with the induction of GAD 6 5 expression upon commencement of the glucose-methanol feed. Cells from the fermentation vessel are analyzed for GAD 6 5 expression as described above for shake flask cultures.

Cells are removed from the fermentation vessel at certain time intervals and subsequently analyzed. Little GAD 6 5 expression is observed during growth on glucose.

Exhaustion of glucose leads to low level expression of the GAD 6 5 protein; expression is enhanced by the addition of MeOH during feeding of the fermentation culture. The addition of methanol has a clear stimulatory effect of the expresion of human GAD 6 5 driven by the methanol-responsive A UGl promoter.

WO 98/02565 PCTIUS97/12581 Example 4 Transformation conditions were investigated to determine the electric field conditions, DNA topology, and DNA concentration that were optimal for efficient transformation of P. methanolica. All experiments used P. methanolica ade2 strain #11.

Competent cells were prepared as previously described. Electroporation was carried out using a BioRad Gene PulserTM.

Three field parameters influence transformation efficiency by electroporation: capacitance, field strength, and pulse duration. Field strength is determined by the voltage of the electric pulse, while the pulse duration is determined by the resistance setting of the instrument.

Within this set of experiments, a matrix of field strength settings at various resistances was examined. In all experiments, the highest capacitance setting (25 gF) of the instrument was used. 100 pl aliquots of electrocompetent cells were mixed on ice with 10 ptl of DNA that contained approximately 1 pg of the ADE2 plasmid pCZR133 that had been linearized with the restriction enzyme Not I. Cells and DNA were transferred to 2 mm electroporation cuvettes (BTX Corp., San Diego, CA) and electropulsed at field strengths of 0.5 kV (2.5 kV/cm), 0.75 kV (3.75 kV/cm), 1.0 kV (5.0 kV/cm), 1.25 kV (6.25 kV/cm), and 1.5 kV (7.5 kV/cm). These field strength conditions were examined at various pulse durations. Pulse duration was manipulated by varying the instrument setting resistances to 200 ohms, 600 ohms, or "infinite" ohms. Pulsed cells were suspended in YEPD and incubated at 30 0 C for one hour, harvested, resuspended, and plated. Three separate sets of experiments were conducted. In each set, electroporation conditions of 0.75 kV (3.75 kV/cm) at a resistance of "infinite" ohms was found to give a dramatically higher transformation efficiency than other conditions tested (see Fig. 1).

After the optimal pulse conditions were established, the influence of DNA topology on transformation efficiency was investigated. Electrocompetent cells were mixed with 1 tg of uncut, circular pCZR133 or with 1 pg of Not I-digested pCZR133. In three separate experiments, an average of roughly 25 transformants were recovered with circular DNA while linear DNA yielded an average of nearly 1 x 104 transformants. These data indicate that linear DNA transforms P. methanolica with much greater efficiency than circular DNA.

Finally, the relationship between DNA concentration and transformation efficiency was investigated. Aliquots of linear pCZR133 DNA (1 ng, 10 ng, 100 ng and 1 pg in pl H20) were mixed with 100 ptl electrocompetent cells, and electroporation was carried out at 3.75 kV/cm and "infinite" ohms. The number of transformants varied from about 10 (1 ng DNA) to 104 (1 pg DNA) and was found to be proportional to the DNA concentration.

Example Integration of transforming DNA into the genome of P. methanolica was detected by comparison of DNA from wild-type cells and stable, white transformant colonies. Two classes of integrative transformants were identified. In the first, transforming DNA was found to have integrated into a homologous site. In the second class, transforming DNA was found to WO 98/02565 PCTIUS97/12581 21 have replaced the endogenous A UG1 open reading frame. While not wishing to be bound by theory, this second transformant is believed to have arisen by a "transplacement recombination event" (Rothstein, Methods Enzymol. 194:281-301, 1991) whereby the transforming DNA replaces the endogenous DNA via a double recombination event.

P. methanolica ade2 strain #11 was transformed to Ade with Asp I-digested pCZR140, a Bluescript® (Stratagene Cloning Systems, La Jolla, CA)-based vector containing the P. methanolica ADE2 gene and a mutant of A UG1 in which the entire open reading frame between the promoter and terminator regions has been deleted (Fig. Genomic DNA was prepared from wild-type and transformant cells grown for two days on YEPD plates at 30 0

C.

About 100-200 pil of cells was suspended in 1 ml H20, then centrifuged in a microcentrifuge for seconds. The cell pellet was recovered and resuspended in 400 pl of SCE DTT zymolyase (1.2 M sorbitol, 10 mM Na citrate, 10 mM EDTA, 10. mM DTT, 1-2 mg/ml zymolyase 100T) and incubated at 37°C for 10-15 minutes. 400 p.l of 1% SDS was added, and the solution was mixed until clear. 300 l.1 of 5 M potassium acetate, pH 8.9 was added, and the solution was mixed and centrifuged at top speed in a microcentrifuge for five minutes. 750 pl of the supernatant was transferred to a new tube and extracted with an equal volume of phenol/chloroform. 600 pl of the resulting supernatant was recovered, and DNA was precipitated by the addition of 2 volumes of ethanol and centrifugation for 15 minutes in the cold. The DNA pellet was resuspended in 50 ml TE (10 mM Tris pH 8, 1 mM EDTA) 100 pg/ml RNAase for about 1 hour at 65 0 C. 10-pl DNA samples were digested with Eco RI (5 p.1) in a 100 pl reaction volume at 37 0 C overnight. DNA was precipitated with ethanol, recovered by centrifugation, and resuspended in 7.5 p TE 2.5 pl 5X loading dye. The entire 10 ml volume was applied to one lane of a 0.7% agarose in 0.5 X TBE (10 X TBE is 108 g/L Tris base 7-9, g/L boric acid, 8.3 g/L disodium EDTA) gel. The gel was run at 100 V in 0.5 X TBE containing ethidium bromide. The gel was photographed, and DNA was electrophoretically transferred to a positively derivatized nylon membrane (Nytran® Schleicher Schuell, Keene, NH) at 400 mA, 20 mV for 30 minutes. The membrane was then rinsed in 2 X SSC, blotted onto denaturation solution for five minutes, neutralized in 2 X SSC, then cross-linked damp in a UV crosslinker (Stratalinker®, Stratagene Cloning Systems) on automatic setting. The blot was hybridized to a PCR-generated AUG1 promoter probe using a commercially available kit (ECLTM kit, Amersham Corp., Arlington Heights, IL). Results indicated that the transforming DNA altered the structure of the AUG1 promoter DNA, consistant with a homologous integration event (Fig. 2).

In a second experiment, P. methanolica ade 2 strain #11 was transformed to Ade with Not I-digested pCZR137, a vector containing a human GAD65 cDNA between the AUG1 promoter and terminator (Fig. Genomic DNA was prepared as described above from wildtype cells and a stable, white, Ade+ transformant and digested with Eco RI. The digested DNA was separated by electrophoresis and blotted to a membrane. The blot was probed with a PCRgenerated probe corresponding to either the A UG] open reading frame or the A UG1 promoter.

WO 98/02565 PCT/US97/12581 22 Results demonstrated that the A UG1 open reading frame DNA was absent from the transformant strain, and that the A UG1 promoter region had undergone a significant rearrangement. These results are consistent with a double recombination event (transplacement) between the transforming DNA and the host genome (Fig. 3).

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.

WO 98/02565 PCT/US97/12581 23 SEQUENCE LISTING GENERAL INFORMATION APPLICANT: Raymond, Christopher K.

Holderman, Susan D.

Vanaja, Erica (ii) TITLE OF THE INVENTION: TRANSFORMATION OF PICHIA METHANOLICA (iii) NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: ZymoGenetics, Inc.

STREET: 1201 Eastlake Avenue East CITY: Seattle STATE: WA COUNTRY: USA ZIP: 98102 COMPUTER READABLE FORM: MEDIUM TYPE: Diskette COMPUTER: IBM Compatible OPERATING SYSTEM: DOS SOFTWARE: FastSEQ Version (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: Parker, Gary E.

REGISTRATION NUMBER: 31,648 REFERENCE/DOCKET NUMBER: 95-37 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 206-442-6673 TELEFAX: 206-442-6678 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 3077 base pairs TYPE: nucleic acid WO 98/02565 24 STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO FRAGMENT TYPE: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: PCTIUS97/12581

CAGCTGCTCT

TCCCCMACAG

TATTGATGCT

CTGTGGGCGT

TGATTCTCTT

GMACGCTGAC

TGCTTGCTTA

CTCCTGTAAA

AAAATTGCTA

AMAAGAATGA

GCAAATCTGT

]TVGCGGGTA

CCATGAGTGT

TTCTTTCCMA

TTCAGAGTC

TAACTGATAA

TATTMCCAA

TGTCGGGATT

GAATATCAAA

TTAGATGAC

CAAGTGTGAT

TCAAMAGGCA

TAAATACTTG

GCTCCTTGAT

GGATTCCAAT

GCAAAAACTT

TCCACACTCC

ATTACCAGTT

CCACGGTTTC

AAGCGGCTMA

GGGCCGATTC

MAGGAGTACT

CGCTGTATGT

TGGTCACCGG

GGAAGCMAGG

TTCCCTCTGG

GTTCGGCT-TT

AAATTMATTT

GTCGAATCMA

TAGCATATTC

T-TAGGTGGTG

ACTGTGATTC

CATATTGACG

GTTTMCCG

ACTGGCATCA

CAAAAAGAGC

TCGTMATTMA

CGGTGCTCAG

TTAGCCGG

TTGCTTTTCA

ATGTAGAAAG

AAATAACTAT

AAAGTGTTTG

GACTTCGAAA

AGGGCTGTAG

CGTAGCCTGC

TTCGATCCGG

TCTAGTTTFTC

CTACCTMATA

CAGCTCGTMA

TCGCTAACMA

CATCTTTAAA

ACAGGCATCA

GCCAACTTGG

TCGAAAATGG

GCTCATTCAA

TTGAGATTGA

AAATCTTCCC

ATTTGATTIM

TGTTATCCTT TTACTTTGMA CTCTTGTCGG

CGGGATTTCC

GTTTMAGTAA

TAATCTCTGT

ATCGGCAMAC

CAGAACTCTA

GCAAATTAAA

GAGCCTAAAA

TMATAAATAA

ACGAGTAGCT

TCTCGGGCTT

GTCGTITCGG

TATTTATTGA

ATGTGCAAGA

TTTGTGTTTT

TCCTTTAGTT

CATCGGAACA

TCGTATGATC

AGACCAGGCT

TGATCCAA

ACATGTTGAC

ATCACCAGAA

GAATGGCATT

CATGAGGTTT

CTGGGCMATA

GTATTGTTTT

,AAMATATCMA

TAGCTATAGG

AAAGCTGTGA

ACAGTGACTA

TGGAACAGTG

CAGTGGTAGA

CCTTTTTTGC

ATGGTTTACG

TCGGTCTCTC

AATATTTGAC

TCTGGAGAAA

MAGATCTCTG

TTCAGMATGG

GTTGMAGCTG

CCAGCAAAGC

GCMATTGCCG

ACTGATGCGT

ACTATTTCAT

GCTGTTGCCG

TTGACIAACTT

TTTCCAMAGG

ATTCGCATTT

CTTATCTT

GGMAGTTTAC

CAAGTAGGMA

TTGGTGACGG

GTACAACMT

GCAGCAGATT

TTTTTCGATA

AAAGTATCAG

ATGTGMATGT

TCCAGCGACC

CCTAAAGATT

CAGCGGCCAG

ACTCGCAAMC

CACACAGATT

AAATCAACGC

MATTGGCTGC

TGGTTGAAGT

TGATCMAAGA

AATCTTGTAG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 WO 98/02565 WO 9802565PCT/US97/12581

TG]TGAAAGT

GCTAAAATCT

ATATATACCT

TCCATFITCA

CTACCCAACT

TAGAG-TTMC

TTTCCCAGGT

AGTCAACGAA

CACCTCGCAA

CACTTGTTTG

GCAAAACGGT

CTTATACGGT

ATCMATGACT

TCTCGAAGAA

TGTCATCAIG

GCAATTAAC

GGCCAAGTAT

TGGTGCCGCT

CCCTGTTAAA

AAGAGGTATT

TATCACAATC

MATATGGAMA

TACTTGAGTA

TTMATTGTTC

TTGAAATTTC

ACMATMTAT

AGATATTGCT

CACAAAAATT

ACACCTAATA

AGCGCAGCAT

AGAACAATGG

GMAGCTTTGA

MAGGAGTTAG

GTTGAAACCA

GATACTGTCC

GCTGGTATCT

ATTGCCCCAA

TTTGAAGCTC

IGGACTCCAT

GAGTTCAAGA

MGACTACAA

GACTGTGAGC

CAGTACACTA

GGTTCCGATT

GTTCCATTTG

GCCATTGATG

CATTTACCGG

GGCTCTACTT

CCTGTTGCTA

TTAGGTGCCG

ATGAAGTTV

CATACMAGAA

TAACTGTTAA

TAGTTGCTCG

ATTA]TVCAA

AGCGCTIATC

TTCCGAACCT

CGGAG

CTTTAGAAGA

CCTATGACGG

MAGTTTTAGA

CTGTVATGGT

TCCACCAAAA

AAAAGAAGGC

TTGGTGTTGA

GACCTCACMA

ATGTTAGGGC

CTACCCMAGC

TGTGTMAAG

GACCAGGCAG

GTAGATTACA

CAGATTCCAT

CGGACCTACC

AAGTCACTAT

CTCCAAAGAG

GMATGGTTGC

TGGAIGGTGT

CIGTGGCTAT

GCGATCCAAA

GGGCAAGGCT

STAGAACCTT

TTTCTGCITT

TAAGAGGAAA

CACTGCTATA

ATTATCCT-TA

AGTTGGTGCC

AAGAGGTMAT

TGACAGGCCG

TGTGAGAICA

CMACATCTGT

CCAMTTTIG

AATGTT1TTA

TTCTGGTCAC

CATTACTGGT

TATTATGITG

AGCACTAGMA

AAAMATGGGT

TIACATAGMA

TCCGGGCACT

AGTCATGTCT

CGTTTCCGCT

AGGGTTGAAG

GGCGATGACG

TGATTCACTA

TAACAATGCT

TACTTGICTG

GAAAAATTGG

TTATAYVTGA

GCATTTCTGA

CTGCATTCA

TGGTAGTTTT

ATTGTTCATC

AAATACGGCT

ITTGTTGTCA

TTATACGCCG

ATCGATGGCC

CACACTGTCT

GCTGACMACG

TTACAAAATG

TATACCATCG

CTACCCATGC

AACGTTTTAG

ACTCCTCATG

CACATTMATA

GGTACGACTA

TCAAGCAAGC

CTAGGTTGIA

CATAGMACCC

TGCATCATTG

C CGCTGCCG

CACTCCATCG

ACTMACGCTG

CMATGGMAGT

MAAATGGTGG

TATAGTACTT

MAAGTTAAG

AATMACATTA,

ATAGGTTTGG

GACGCAAATC

TCCCATACAT

AAGACMAGTC

AGAAATGGGC

MAG1TVATTC

TTGCTCCAGC

CTGTCAAATC

GTGACTIATI

ACGCTTGTGT

CGAAGAACTT

GTGGCGATGA

CTTCIGTTTA

TAGTTTCTCA

ACAGCATCCC

CATTAGTCGG

ATATATTGMA

CACAAAGAAT

CTGGTGCTGG

TTATIGGIGI

TTCAAATGCC

CCTTGCTAGC

TTATATGAAC

ATATGMAGM

ACTCAMAGTC

ACMAGAAATC

ACAATAAATG

TTAGGATTVG

GACGCATTTC

1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3077 GITTTTCACT ICICCAGAIC TIGGITTAGI ATAGCTTTTG WO 98/02565 26 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 3386 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO FRAGMENT TYPE: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: PCTIUS97112581

GAATTCCTGC

MGTGTAGTA

CCCTAATCTG

GGGGCAGCAT

MGTTTTATC

AAACTCAMAT

GTTTTGTTMA

ACACTTTCCT

TGTGGGGACA

CAAGACIAAA

AGGCAGGCAG

TTCAMATAAC

GATCCATCAC

GCTCCTTCAA

MAGITITAT

CCGCTCTCTC

CTCCGTGTAC

TTCTTTTGAA

TCAACCTTGT

TTACCAATTA

AGCCCGGGGG

GCCGGACTGA

TTMACAGTCT

CGAGACTCGA

TG11TTTAGA

TMTGGAAAT

ATGACTATCG

GCTTGCTGGT

CTTGACATCT

CAACCCTTTG

GCAGGCAGCG

AGCCTGCTGC

CTTTTGTCGT

TAATT6AATC

CTCTATGGCC

TTTACCCCAG

MAGCGGAGCT

ACTCTTGGTC

TCAACATTCT

ATCAATCTTC

ATCGGGTAGT

MAGGTTIIAG

CGGAGTATAC

GATGGTACAT

ATTIAAAMGAC

AGCCTGTTTT

MACAAGCCAT

TGACTCTCCT

CACATGCACC

TCCTGCTCTT

GGCTGCCTGC

TATCTGTGAC

ACTCCGACAA

AAATAAGCAT

MACGGATAGT

ATTCTCMAAG

TTTGCCTCCC

AAATCAAATC

ATAAATCGAT

AAATTTCAAA

GGAATGCACG

GAGTCTGTT

AAAAAAGTMA

ACTTAAAAGC

GATTGTTGTA

GMAAAATACA

GAAATAGCAC

CATACAAACA

CCAGATTMAT

TTCTTTCTCA

CATCTCTMAT

CAGATTGGGA

TGATCCTTCC

AMTAGTAAA

CTATCTGCTT

CTMATATCTG

ATCCTCTTGC

AAA.CAAAACC

ATAAATATMA

TATTTTCTAC

GTTATACCCA

GTTTGTTCAT

GTCAAMTATC

TGCCATATTG

ACAACGIT

CCTTCTTAAG

ATTTCTGCCA

CCCAMAAGGG

TTCCCCAGAC

CACCGCGTGG

CGCTGCTCCT

CACCCCCCTC

CTGTCATCTT

ATCGCATACA

MATTCCATCC

CCCCTTGTCT

TTTGTTTCGG

AAACCTTCTA

CCTTATCCCT

TTGCTTTATT

CTCCIAAATMA

GTGCATCATT

AAGGTGGCCG

AGGMACTTCA

GTGCCTACAT

TACTOACAMA

GTCACTTTTA

AMACTTTCAG

GATGCGGAGA

GTGTGT.rGCGC

CCCCCCTGGC

CCCTCCGAAT

CTGGCAATCA

AACGTCATGA

ACTTTGGGAA

ATTGTCCTTT

TTATTTTTT

TTCCATCAGA

CCCTTGTTTT

ACTCAGTATT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 WO 98/02565 WO 9802565PCTIUS97/12581

AACATTTGTT

]TTCAA]TVA

TACGAAAA

TATTGTTGTC

CGAMAACGTC

TTACY FAGCA

CTCTTCAAGA

CTTGGGTGGT

CGATGATTGG

TGAMACTTAT

GGTTTCATTT

TCAAGGTATT

CTGGTTGMAG

TCACCCAACC

GATTATCATT

TCTCCAAAG

TGGTACTAIC

GAGACMAGTT

TCACTACTGT

TGTTAGAGGI

TGGTCCAIIA

ATTAGCCACT

AGAIMAGCCA

TCCAAACGGT

CGTTCACGT

CGATCCAAGA

AAGMATGGAC

ACCAGCCAGA

CTTTACTGCT

TAAACCMACI

CAICTITATI

AAATCTTACT

GGTGGTGGTT

ACAGTTGCTT

GGIGTTTATC

CCATCACCAC

GGTTCTTCCA

GAATCTGMAG

CAAAGACCAT

GGTMACTAIA

CCATTTGTTG

TGGATCAACA

ATGAGAAACA

GAAAACGGTG

ACCCMAGTTG

TCAICACCAT

GGTAITAMAC

TTCTTCACTC

GATAMAGCTG

ACCACTAATG

GCTGATGACG

TTAATGCACI

AAGTACATGT

GTTTCTCCAA

GATATGTGGC

TG]TVTGCCG

GCTGCTGACA

MACTTGTACC

ATAACITTTA

TATTAACGMA

ATTMATTTCT

CCACCGGTTG

TMATCGAAGG

CAAGMAACAI

ACTTGAACGG

TCAACTTFCTT

GTTGGACIAC

GTMACMCAG

CTTATCCAAA

ATGATGCIGA

GAGACTTAGG

AGCAAMACTT

ITGCTACIGG

CIAGMACTTT

TAGTTTIGCA

CAATTGTTGA

CATACCATGT

TTCAAAAATC

GTATTGAGGC

AA1TCAGAGC

ACTCTCTMAT

GCATGTTCCA

ACCCATACGA

CAATGGTITG

GTGAAGTTAC

TGGACTTGGA

ACGGTTCATG

ACTGGCTTTA

ATCTTACGA

CAAAATGGCT

TGCTCTTGCT

TGGTGMAAAC

GAGATTAGAC

TAGAAGAGCT

GATGTACACC

CGATGMATTA

AGMATTGCAC

CGGTCAAGAT

AGATTGAAA

TAGMAGATCC

GTTCTTGATIF

TGTTMAGACT

CMAGGCTAGA

AAGATCTGGT

CTTACCAGGT

CAAGCCAGAT

TGCITTCGAC

AGGTGTIMAG

TGCTTATGAT

TVCTGGTTC

CTTCTTGGMA

TGCTCCTGAC

GTCTTACMAG

ITCTCACCAC

AACTACTA

GACTGTTCCA

GAAGTTTAT TTAACAICAG

ATTMCTCAA

ATTCCAGATG

GGTAGA1TAG

MACATCAACA

ICPAAGACTG

ATTGTTCCAT

AGAGCCTCTG

TTACCACTAA

GGTTTCGATG

TICATTAGAG

TGTTCCCACG

GATTCIGCTC

ACTTCCACCA

GTTCCAATGA

I4AGCAAATTA

ATCGGTTCCG

GTTGGTATGA

ACTCCATCAT

CAATGGTATG

ATTAGACCAA

GACTACTTTG

TTTGGTGACC

TATCCATTCT

]TVGATCCAG

MGTCCAGAG

CCACACTACC

GCTTATGCTG

ATTGAAMAGC

TCAAMCTTI

AATTGAIAT

GTAACTTGGA

ACCCATGGGT

CTACTF[TA

GTGCTMACAT

CCTCCGATTA

TGAAGAAGAT

GTCCMATTMA

CTGCCGMATC

GTGCTGAGCA

ATGCTIACAT

AGTGTGAAPA

AGCCAACIGG

TTGTTTCTTG

CTCACAAGTT

ACTTCCMAGA

ICGATGACTT

CTAACAAGGA

CTGMAGMGA

GTAACAAGCC

ACACCAAGAT

CCAGAGGTTT

GTTTCATGAA

AMACTGCCAG

CATACGACIC

GTGCAGACCA

CMCTCCAAA

1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 WO 98/02565 PCT/US97/12581

GAACGCTGCT

GGAGGATGAT

TCTTGGTACT

TGTTGACTCC

TTGCCCAGAT

TTCTACCTTA

AAACTTCAAA

TGGATATTTT

CACGTTACTT

GCTGCTATCG

TGTTCAATGG

AGATTAAACG

AATGTTGGTT

GTTGCTGAAG

TTGGGTACTT

TATAATTTTT

CTAACCAAGT

AAGATTACAT

CTCCAAGAGA

TTTACGGTGT

GTAACACTTA

ACTTGGGCTA

ATGAAGAAGC

GAGAGT

TGAAAAACAT

CAGAGAACAC

AGGTTCTAAG

TGAAAAGTTG

CTCTACTGCT

CTCTGGTGAT

TGGTCTAGCT

CGTGACATCG

ACTGAAACCA

GTTGTCCCAA

AAGGTTGCTG

TTGTTAATCG

GCTTTGAAGA

AGATTCTAGG

AATACACCAA

CATGGCATTG

CTGGTGGTGT

ATTTATCAAT

GTGAAAAGGC

TGACTGTTCC

GCTGCCTGTT

3000 3060 3120 3180 3240 3300 3360 3386 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TGATCACCTA GGACTAGTGA CAAGTAGGAA CTCCTGTA INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CAGCTGCCTA GGACTAGTTT CCTCTTACGA GCAACTAGA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 98/02565 29 (xi) SEQUENCE DESCRIPTION: SEQ ID TGGTTGAAGT GGATCAA INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GTGTGGTCAC CGAAGAA PCT/US97/12581

Claims (4)

1. A method for introducing a DNA molecule into a Pichia methanolica cell, comprising exposing a Pichia methanolica cell, in the presence of a linear DNA molecule, to an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm and a time constant of from 1 to 40 milliseconds, whereby said DNA molecule is introduced into said cell.

2. A method according to claim 1 wherein said DNA molecule comprises a segment encoding a polypeptide, other than a P. methanolica polypeptide, operably linked to a P. methanolica gene transcription promoter and a P. methanolica gene transcription terminator.

3. A method according to claim 2 wherein said transcription promoter is a P. methanolica A UGI gene promoter.

4. A method according to claim 3 wherein said AUG1 gene promoter comprises a sequence of nucleotides as shown in SEQ ID NO:2 from nucleotide 24 to nucleotide

1354. A method according to claim 2 wherein said DNA molecule further comprises a selectable marker gene that complements a mutation in said cell. 6. A method according to claim 5 wherein said selectable marker gene is a P. methanolica gene. 7. A method according to claim 5 wherein said marker gene is a P. methanolica ADE2 gene. 8. A method according to claim 7 wherein said ADE2 gene comprises a sequence of nucleotides as shown in SEQ ID NO:1 from nucleotide 407 to nucleotide 2851. 9. A method according to claim 1 wherein said DNA molecule comprises a selectable marker gene that complements a mutation in said cell. A method according to claim 9 wherein said marker gene is a P. methanolica gene. 11. A method according to claim 9 wherein said marker gene is a P. methanolica ADE2 gene. 12. A method according to claim 11 wherein said ADE2 gene comprises a sequence of nucleotides as shown in SEQ ID NO:1 from nucleotide 407 to nucleotide 2851. 13. A method according to claim 1 wherein said time constant is from 15 to 30 milliseconds. 14. A method for introducing a DNA molecule into a Pichia methanolica cell, substantially as hereinbefore described with reference to any one of the Examples. A method for transforming P. methanolica with heterologous DNA, comprising exposing a population of P. methanolica cells, in the presence of heterologous linear DNA molecules, to an o exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5kV/cm and a time constant of from 15 to 40 milliseconds, whereby said heterologous DNA is introduced into at least a portion of said population, and recovering cells into which said DNA has been introduced. 16. A method according to claim 15 wherein from 103 to 105 cells are recovered per microgram of heterologous DNA. 17. A method according to either claim 15 or claim 16 wherein from 0.9 x 104 to 1.1 x 104 cells are recovered per microgram of heterologous DNA. 18. A method according to any one of claims 15 to 17 further comprising the step of recovering integrative transformants from said recovered cells. 19. A method according to claim 18 wherein said recovering step comprises culturing said 20 cells in a growth medium comprising sorbitol as a carbon source. *20. A method according to claim 15 wherein said population of cells is in early log phase S growth. 21. A method according to claim 15 wherein said time constant is from 15 to milliseconds. 25 22. A method for transforming P. methanolica with heterologous DNA, substantially as hereinbefore described with reference to any one of the Examples. 23. A Pichia methanolica cell transformed by the method of any one of claims 1 to 22. Dated 15 February, 2000 ZymoGenetics, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON