Definitions

• 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, Mvcologia 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/Technoloev 1 1 :905, 1993.
• methylotrophic yeasts as hosts for use in recombinant production systems has been slow, due in part to a lack of suitable materials (e.g., promoters, selectable markers, and mutant host cells) and methods (e.g., transformation techniques).
• suitable materials e.g., promoters, selectable markers, and mutant host cells
• methods e.g., 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).
• the present invention provides methods for introducing DNA molecules into Pichia methanolica cells and cells transformed according to these methods.
• the methods comprise 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 the DNA molecule is introduced into the cell.
• 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.
• the transcription promoter is a P. methanolica A UGl 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 rrutation in the host cell.
• the selectable marker gene is a P. methanolica gene, such as a P. methanolica ADE2 gene.
• 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 4.5 kV/cm and a time constant of from 1 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.
• from 10 ⁇ to 10 ⁇ cells are recovered per microgram of heterologous DNA.
• from 0.9 x 10 ⁇ to 1.1 x 10 ⁇ cells are recovered per microgram of heterologous DNA.
• 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.
• the population of cells is in early log phase growth.
• the present invention provides Pichia methanolica cells transformed by the methods disclosed above.
• 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.
• 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.
• 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.
• 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 indicates that DNA 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.
• 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.
• 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.
• 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.
• a selectable marker allows the transformed cells to grow under conditions in which untransformed cells cannot multiply ("selective conditions").
• 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.
• a preferred selectable marker of this type for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5- 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' non- coding sequence, with the initiation ATG codon at nucleotides 1007-1009.
• 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.
• 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.
• host cells in which both methanol utilization genes (AUG1 and AUG2) are deleted.
• host cells deficient in vacuolar protease genes PEP4 and PRB1 are preferred.
• Gene- deficient 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.
• 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 (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 .
• 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 (e.g., Neumann et al., EMBO 11:841-845, 1982) and fungal (e.g., 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.
• 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 4.5 kV/cm and a time constant ( ⁇ ) of from 1 to 40 milliseconds.
• the time constant ⁇ is defined as the time required for the initial peak voltage V 0 to drop to a value of V 0 /e.
• 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 (e.g., BioRad Laboratories, Hercules, CA).
• DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation.
• the DNA molecules will include, in addition to the selectable marker disclosed above, an expression casette comprising a transcription promoter, a DNA segment (e.g., 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. methanolica gene.
• a preferred promoter is that of a P. methanolica alcohol utilization gene (AUG1).
• the initiation ATG codon is at nucleotides 1355-1357.
• Nucleotides 1-23 of SEQ ID NO:2 are non-AUGl 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, A UG2, 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.g., E. coli).
• an alternate host e.g., E. coli.
• the expression segment 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 from other sequences (e.g., 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.g.. Not I).
• 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 .
• 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
• hormones such as insulin, leptin, and glucagon .
• 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 is YEPD (Table 1).
• 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°C.
• 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.
• solid media preferably solid YEPD.
• single colonies from a fresh plate are used to inoculate the desired volume of rich culture media (e.g., YEPD) to a cell density of about 5 - 10 x 10 5 cells/ml.
• Cells are incubated at about 25 - 35°C, preferably 30°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 ⁇ -mercaptoethanol (BME), to reduce cell wall proteins to facilitate subsequent uptake of DNA.
• DTT dithiothreitol
• BME ⁇ -mercaptoethanol
• a preferred incubation buffer in this regard is a fresh solulion of 50 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°C for about 5 to 30 minutes, preferably about 15 minutes.
• the cells are then harvested and washed in a suitable electroporation buffer, which is used ice-cold.
• suitable buffers include pH 6-8 solutions containing a weak buffer, divalent cations (e.g., Mg "1-1" , Ca " * " " ) and an osmotic stabilizer (e.g., a sugar).
• divalent cations e.g., Mg "1-1" , Ca " * " "
• an osmotic stabilizer e.g., a sugar
• a preferred electroporation buffer is STM (270 mM sucrose, 10 mM Tris, pH 7.5, 1 mM MgCl2).
• STM 270 mM sucrose, 10 mM Tris, pH 7.5, 1 mM MgCl2.
• 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 ⁇ l) 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 ⁇ g of DNA (vol. ⁇ 10 ⁇ l). 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 time constant of from 1 to 40 milliseconds, preferably 10-30 milliseconds, more preferably 15-25 milliseconds, most preferably about 20 milliseconds, with exponential decay.
• a pulsed electric field of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant of from 1 to 40 milliseconds, preferably 10-30 milliseconds, more preferably 15-25 milliseconds
• the actual equipment settings used to achieve the desired pulse parameters will be determined by the equipment used.
• resistance is set at 600 ohms or greater, preferably "infinite” resistance, and capacitance is set at 25 ⁇ F to obtain the desired field characteristics.
• capacitance is set at 25 ⁇ F to obtain the desired field characteristics.
• the cells are diluted approximately 10X into 1 ml of YEPD broth and incubated at 30°C for one hour.
• the cells are then harvested and plated on selective media.
• the cells are washed once with a small volume (equal to the diluted volume of the electroporated 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.
• IX yeast nitrogen base 6.7 g/L yeast nitrogen base without amino acids; Difco Laboratories, Detroit, MI
• Cells having an ade2 mutation 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 1 ).
• 250 ⁇ l aliqouts of cells are plated on 4 separate ADE D or ADE DS plates to select for Ade + cells.
• 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 extrachro osomally.
• ARS autonomously replicating sequences
• integrative transformants are generally preferred for use in protein production systems. Integrative transformants have a profound growth advantage over ARS transformants on selective media containing sorbitol as a 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 AUG] 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.
• ADE DS plates are more selective for stable transformants, which form large ( «5 mm) colonies in 5-7 days, while unstable (ARS-maintained) colonies are much smaller ( «1 mm).
• the more selective ADE DS media is therefore preferred for the identification and selection of stable transformants.
• 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.
• transforming DNA segments e.g., promoter, terminator, heterologous DNA, and selectable marker sequences
• 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 (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.
• P. methanolica transformants that carry an expression cassette comprising a methanol-regulated promoter (such as the A UGl promoter) are grown in the presence of methanol and the absence of interfering amounts of other carbon sources (e.g., glucose).
• 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 ⁇ g/L biotin, and 0.56 g/L of -Ade -Thr -Trp powder.
• 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.
• cells are cultivated for two days on minimal methanol plates as disclosed above at 30°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 ⁇ g/L biotin) at a cell density of about 1 x 10 ⁇ cells/ml.
• minimal methanol media 6.7 g/L yeast nitrogen base without amino acids, 10 g/L methanol, 0.4 ⁇ g/L biotin
• Cells are grown at 30°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).
• 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°C for two days. Table 2 Viable Cells
• An amount of cell suspension sufficient to give an ODg ⁇ O 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 ⁇ g/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 ODgQO 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°C for 6 hours.
• nystatin enrichment had decreased the number of viable cells by a factor of 10 ⁇ .
• mutant #3 gave Ade + colonies when mated to #2, complementation testing was repeated with mutant #3. 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 #3. Results of the crosses are shown in Table 3.
• a P. methanolica clone bank was constructed in the vector pRS426, a shuttle vector comprising 2 ⁇ 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 in E. coli MCI 061 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- ⁇ l aliquots were plated onto ADE D, ADE DS, URA D and URA DS plates (Table 1). Plates were incubated at 30°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.
• Total DNA was isolated from the HBY21A transformants Adel and Ade6 and used to transform E. coli strain MCI 061 to Amp R .
• DNA was prepared from 2 Amp R colonies of Adel and 3 Amp R colonies of Ade6. The DNA was digested with Pst I, Sea I, and Pst I + Sea I and analyzed by gel electrophoresis. All five isolates produced the same restriction pattsrn.
• 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 NO:3) was designed to prime at bases 406-429 of the ADE2 DNA (SEQ ID NO:l), 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 ⁇ l 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 ⁇ l Taq polymerase (Boehringer Mannheim).
• PCR was run for 30 cycles of 30 seconds at 94°C, 60 seconds at 50°C, and 120 seconds at 72°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 Sai l.
• Vector pRS426 was digested with Spe I and treated with calf intestinal phosphatase.
• Four ⁇ l of PCR fragment and 1 ⁇ l of vector DNA were combined in a 10 ⁇ l 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 pCZRl 18.
• 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.
• 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, 1 1, and 12 were pooled.
• the cells were then harvested and resuspended in 200 ml of ice- cold STM (270 mM sucrose, 10 mM Tris, pH 7.5, 1 mM MgC ? )- The cells were harvested and resuspended in 100 ml of ice-cold STM. The cells were again harvested and resuspended in 3-5 ml of ice-cold STM. 100- ⁇ l aliquouts of electrocompetent cells from each culture were then mixed with Not I-digested pADEl-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 1000 ⁇ resistance and capacitance of 25 ⁇ F. After being pulsed, the cells were diluted by addition of 1 ml YEPD and incubated at 30°C for one hour. The cells were then harvested by gentle centrifugation and resuspended in 400 ⁇ l minimal selective media lacking adenine (ADE D). The resuspended samples were split into 200- ⁇ l aliqouts and plated onto ADE D and ADE DS plates. Plates were incubated at 30°C for 4-5 days. Mutants #6 and #1 1 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.
• the P. methanolica clone bank disclosed in Example 1 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 ⁇ l 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 hoidini, and Pichia pastoris.
• the amplification reaction was run for 30 cycles of 94°C, 30 seconds; 50°C, 30 seconds; 72°C, 60 seconds; followed by a 7 minute incubation at 72°C.
• One pool (#5) gave a -600 bp band.
• DNA sequencing of this PCR product revealed that it encoded an amino acid sequence with -70% sequence identity with the Pichia pastoris alcohol oxidase encoded by the AOX1 gene ⁇ ind about 85% sequence identity with the Hansenula polymorpha alcohol oxidase encoded by the MOX1 gene.
• the sequence of the cloned A UGl 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.
• Example 3 ade2 mutant P. methanolica cells are transformed by electroporation essentially as disclosed above with an expression vector comprising the AUGl promoter and te ⁇ ninator, human GAD65 DNA ( arlsen 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 BactoTM-agar (Difco), 6.7 g/L yeast nitrogen base without amino acids (Difco), 10 g/L methanol, and 0.4 ⁇ g/L biotin.
• agar minimal methanol plates (10 to 100 colonies per 100-mm plate) containing 20 g/L BactoTM-agar (Difco), 6.7 g/L yeast nitrogen base without amino acids (Difco), 10 g/L methanol, and 0.4 ⁇ g/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°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 30 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, 5% powdered nonfat milk) for 30 minutes at room temperature.
• TTBS-NFM 20 mM Tris pH 7.4, 0.1% Tween 20, 160 mM NaCl, 5% powdered nonfat milk
• 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 20, 160 mM NaCl) two times for five minutes each.
• the filters are then incubated in goat anti- mouse antibody conjugated to horseradish peroxidase (1 ⁇ g/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.
• candidate clones are cultured in shake flask cultures. Colonies are grown for two days on minimal methanol plates at 30°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, 0.4 ⁇ g/L biotin) at a cell density of 1 x 10 ⁇ cells/ml. The cultures are grown for 1-2 days at 30°C with vigorous shaking. 0.2 ml of 50% methanol is added to each culture daily.
• minimal methanol media 6.7 g/L yeast nitrogen base without amino acids, 10 g/L methanol, 0.4 ⁇ g/L biotin
• 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 ⁇ g/ml leupeptin, 1 ⁇ g/ml pepstatin, 1 ⁇ g/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 (5% 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.
• sample buffer 5% SDS, 8 M urea, 100 mM Tris pH 6.8, 10% glycerol, 2 mM EDTA, 0.01% bromphenol glue
• 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 conditions.
• Cells are first cultivated in 0.5 liter of YEPD broth at 30°C for 1 - 2 days with vigorous agitation, then used to inoculate a 5-liter fermentation apparatus (e.g., BioFlow III; New Brunswick Scientific Co., Inc., Edison, NJ).
• the fermentation vessel is first chaiged with mineral salts by the addition of 57.8 g (NH4)2SO4, 68 g KH 2 PO4, 30.8 g MgSO4-7H2O, 8.6 g CaSO4-2H2O, 2.0 g NaCl, and 10 ml antifoam (PPG).
• H2O is added to bring the volume to 2.5 L, and the solution is autoclaved 40 minutes. After cooling, 350 ml of 50% glucose, 250 ml 10 X trace elements (Table 4), 25 ml of 200 ⁇ g/ml biotin, and 250 ml cell inoculum are added.
• the fermentation vessel is set to run at 28°C, pH 5.0, and >30% dissolved O2.
• 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.
• a glucose-methanol feed supplemented with NH + and trace elements is delivered into the vessel at 0.2% (w/v) glucose, 0.2% (w/v) methanol for 5 hours followed by 0.1% (w/v) glucose, 0.4% (w/v) 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 GADg5 expression upon commencement of the glucose-methanol feed. Cells from the fermentation vessel are analyzed for GADg5 expression as described above for shake flask cultures.
• 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 ade 2 strain #1 1. Competent cells were prepared as previously described. Electroporation was carried out using a BioRad Gene PulserTM.
• Field strength is determined by the voltage of the electric pulse, while the pulse duration is determined by the resistance setting of the instrument.
• the highest capacitance setting (25 ⁇ F) of the instrument was used. 100 ⁇ l aliquots of electrocompetent cells were mixed on ice with 10 ⁇ l of DNA that contained approximately 1 ⁇ g 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°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 ).
• P. methanolica ade 2 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 AUGl in which the entire open reading frame between the promoter and terminator regions has been deleted (Fig. 2).
• Genomic DNA was prepared from wild-type and transformant cells grown for two days on YEPD plates at 30°C. About 100-200 ⁇ l of cells was suspended in 1 ml H2O, then centrifuged in a microcentrifuge for 30 seconds.
• the cell pellet was recovered and resuspended in 400 ⁇ l 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 ⁇ l of 1% SDS was added, and the solution was mixed until clear. 300 ⁇ l 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 ⁇ l of the supernatant was transferred to a new tube and extracted with an equal volume of phenol/chloroform.
• SCE + DTT + zymolyase 1.2 M sorbitol, 10 mM Na citrate, 10 mM EDTA, 10 mM DTT, 1-2 mg/ml zymolyase 100T
• 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 ⁇ g ml RNAase for about 1 hour at 65°C. 10- ⁇ l DNA samples were digested with Eco Rl (5 ⁇ l) in a 100 ⁇ l reaction volume at 37°C overnight. DNA was precipitated with ethanol, recovered by centrifugation, and resuspended in 7.5 ⁇ l TE + 2.5 ⁇ l 5X loading dye.
• X TBE is 108 g/L Tris base 7-9, 55 g/L boric acid, 8.3 g/L disodium EDTA
• 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® N+, 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 (Stratal inker®, Stratagene Cloning Systems) on automatic setting.
• the blot was hybridized to a PCR-generated AUGl 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 AUGl promoter DNA, consistent with a homologous integration event (Fig. 2).
• P. methanolica ade 2 strain #11 was transformed to Ade + with Not I-digested pCZR137, a vector containing a human GAD65 cDNA between the AUG] promoter and terminator (Fig. 3).
• Genomic DNA was prepared as described above from wild- type cells and a stable, white, Ade + transformant and digested with Eco Rl. The digested DNA was separated by electrophoresis and blotted to a membrane. The blot was probed with a PCR- generated probe corresponding to either the AUGl open reading frame or the AUGl promoter.
• Results demonstrated that the AUGl open reading frame DNA was absent from the traisformant strain, and that the AUGl 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).
• MOLECULE TYPE Genomic DNA
• HYPOTHETICAL NO
• ANTISENSE NO
• FRAGMENT TYPE (vi) ORIGINAL SOURCE:
• MOLECULE TYPE Genomic DNA
• HYPOTHETICAL NO
• ANTISENSE NO
• FRAGMENT TYPE (vi) ORIGINAL SOURCE:
• GAATTCCTGC AGCCCGGGGG ATCGGGTAGT GGAATGCACG GTTATACCCA CTCCAAATAA 60
• CAAGACAAAA CAACCCTTTG TCCTGCTCH ⁇ CT ⁇ CTCA CACCGCGTGG GTGTGTGCGC 600
• AAAGTTTTAT CTCTATGGCC AACGGATAGT CTATCTGCTT AATTCCATCC ACTTTGGGAA 900
• CTCCGTGTAC AAGCGGAGCT TTTGCCTCCC ATCCTC ⁇ GC TTTGT ⁇ CGG TTA ⁇ T ⁇ TT 1020 ⁇ CTTTTGAA ACTCTTGGTC AAATCAAATC AAACAAAACC AAACC ⁇ CTA TTCCATCAGA 1080
• TTTCAATTTA CATCTTTA ⁇ TA ⁇ AACGAA ATCTTTACGA ATTAACTCAA TCAAAACTTT 1320
• CTTTACTGCT AACTTGTACC ACGG ⁇ CATG GACTG CCA ATTGAAAAGC CAACTCCAAA 2940 GAACGCTGCT CACGTTACTT CTAACCAAGT TGAAAAACAT CGTGACATCG AATACACCAA 3000

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• 1997
  • 1997-07-14 EP EP97935001A patent/EP0920525A1/de not_active Withdrawn

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