MXPA00002567A - Chromosomal mutagenesis in pichia methanolica - Google Patents

Abstract

Methods for altering a selected chromosomal locus in i(P. methanolica) cells and cells comprising such altered loci are disclosed. A linear DNA construct comprising (i) a segment comprising a portion of the target locus in which at least one nucleotide pair is altered and (ii) a selectable marker that complements adenine auxotrophy is introduced into cells auxotrophic for adenine. The cells are cultured under selective conditions, and cells in which the linear DNA construct has been chromosomally integrated by homologous recombination are identified. The cells are then cultured under conditions whereby cells auxotrophic for adenine can be identified, and a subset of such cells in which the altered locus has been chromosomally integrated are identified.

Description

CHROMOSOMY MUTAGENESIS IN PICHIA METHANOLICA BACKGROUND OF THE INVENTION Methylotrophic yeasts are those yeasts that are capable of using methanol as the sole source of carbon and energy. Yeast species that have the biochemical pathways necessary for the use of methanol are classified into four genera Eansen ul a, Pi chi a, Candi da, and Torul opsi s. 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 8 _: 72-76, 1992). Additionally, not all species within those genera are capable of using methanol as a source of carbon and energy. As a consequence of this classification, there are large differences in physiology and metabolism between the individual species of a genus. The ilotrophic met yeasts are. attractive candidates for use in recombinant protein production systems. Some methylotrophic yeasts have been shown to grow rapidly to a REF; 33094 higher biomass in a defined minimum medium. Certain genes of ilotrophic yeasts are tightly regulated and highly expressed under induced or de-repressed conditions, suggesting that the promoters of these genes may be useful for producing commercially valuable polypeptides. 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. The development of ilotrophic metal yeasts as hosts for use in recombinant protein 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). The most highly developed metalotrophic host systems use Pi ch ia pa sori s and Hansen ul a polymorpha (Faber et al., Curr. Genet, 2_5: 305-310, 1994, Cregg et al., Ibid, Romanos et al. , ibid., United States 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) More recently, they have been developed ( IPO Publication WO 9717450) materials and techniques useful for producing foreign proteins in Pichia methanolica. However, a need remains in the art for additional techniques that can be used to manipulate the P. methanolica genome to extend the understanding of this organism and produce strains that can be used in large-scale protein production systems. A necessary tool is a technique for the directed mutagenesis of P. Methanolica. The directed utagénesis allows the introduction of mutations in the predetermined genomic loci, allowing the selective alteration of the gene activity. Useful alterations include, for example, mutation of promoter sequences to increase gene expression, introduction of heterologous genes into particular sites, and generation of protease auxotrophies and deficiencies. The techniques developed for the germination yeast Saccharomyces cerevisiae are inadequate for P. methanolica. For example, the "enter / exit" method developed by Scherer and Davis (Proc. Nati, Acad. Sci. USA 7_6: 1035, 1979) and summarized by Rothstein (Methods Enzymol., 194: 28.1, 1991) requires a selection against the presence of the URA3 marker, such as by the addition of 5-FOA (5-fluoro-orotic acid) to the culture medium. This method is inadequate with P. methanolica because the cells are not resistant to 5-fluoro-orotic acid (5-FOA), and a URA marker is not available for P. methanolica. The present invention provides methods for producing mutations and directed in the genome of P. methanolica, cells having these mutations, and others, related advantages.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a method for altering a chromosomal locus of Pichia methanolica cells, comprising the steps of: (a) selecting an objective chromosomal locus of the cells; (b) providing a population of P. methanolica cells each comprising a chromosomal copy of the selected target locus, wherein the cells are auxotrophic for adenine; (c) introducing into the cells a linear DNA construct comprising (i) a segment comprising a portion of the target chromosomal locus in which at least one pair of nucleotides is altered and (ii) a selectable marker that complements the auxotrophy of adenine; (d) culturing the cells of step (c) under conditions that are selective for the presence in the cells of the selectable marker; (e) identifying a subset of the cultured cells in which the DNA construction segment and the selectable marker have been chromosomally integrated by homologous recommendation, resulting in a tandem duplication of the target chromosomal locus; (f) culturing the identified subset of cells under conditions where the protophic rheumatic cells for adenine grow and exhibit a first phenotype, and auxotrophic cells for adenine grow and exhibit a second phenotype; (g) recovering cells that are auxotrophic for adenine; and (h) identifying a subset of the auxotrophic cells in which the segment of the DNA construct has been chromosomally integrated, whereby the target chromosomal locus is altered. Within one embodiment of the invention, a plurality of nucleotide pairs of the portion of the chromosomal locus is altered. Within a related mode, from 1 kbp to 2 kbp of the portion of the chromosomal locus is altered. Within another embodiment, the alteration is a deletion of at least one pair of nucleotides. Within additional embodiments, the target chromosomal locus encodes a protease, such as proteinase A or proteinase B, an alcohol oxidase, or a nutritional marker. Within the method described above, steps (a) through (h) can be repeated, whereby two or more chromosomal loci are altered. Within certain embodiments of the invention, a chromosomal locus encoding a protease and a second chromosomal locus encoding an oxidase are altered. The invention also provides a Pi chi cell that is produced by the method described above. These and other aspects of the invention will become apparent with reference to the following detailed description and the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates one embodiment of the invention, by which a gene expression is introduced into the chromosomal locus. Figure 2 shows a partial restriction map of an alcohol oxidase (AUG1) gene of P. m e than ol i ca. The open arrow indicates the open reading frame. The locations of the original PCR product, sequenced region, and its gene pressure are shown. Figure 3 shows a partial restriction map of a genomic clone comprising a PEP4 gene of P. me tha n ol i ca. The PCR product used to identify the gene is shown as complementary half-arrows. A fragment of 420 bp was sequenced to the left of the Asp718 site. Was the pep4 allele created? by deleting the indicated region between the BamHI and Ncol sites. Figure 4 shows a partial restriction map of a genomic clone comprising a PRB1 gene of P. me than ol i ca. The PCR product used to identify the gene is shown as complementary half-arrows. The prbl allele? was generated by deleting the indicated region between the Ncol and EcoRV sites. Figure 5 illustrates a partial restriction map of the second alcohol oxidase (AUG2) gene of P. me than ol i ca. The open arrow indicates the open reading frame. The position of the terminator codon was estimated based on the lengths of other known alcohol oxidase coding regions. The locations of the original PCR product, the sequenced region and the gene deletion are shown. Figure 6 illustrates the plasmid pCZRl34. Figure 7 illustrates the plasmid pCZRl40-6.

DETAILED DESCRIPTION OF THE INVENTION A "chromosomal locus" is a region of DNA found in the genome of a cell. A chromosomal locus may comprise, but may not need, one or more genes. A "DNA construct" is a DNA molecule, either single or double strand that has been modified through human intervention to contain segments of DNA combined and juxtaposed in an arrangement that does not exist in nature. "Homologous recombination" is a genetic recombination between pairs of DNA molecules that have regions of sequence identity. "Linear DNA" denotes DNA molecules that have free 5 'and 3' ends, which are non-circular DNA molecules. Linear DNA can be prepared from closed, circular DNA molecules, such as plasmids, by enzymatic digestion or physical disruption.

A "nutritional marker" is a gene that codes for a protein required for the biosynthesis of a necessary nutrient. Nutritional markers include genes that code for enzymes. required for the biosynthesis of amino acids and nucleotides. The term "operably linked" indicates that the DNA segments are arranged, so that they function according to their intended purposes, for example, the transcription starts at the promoter and proceeds through the coding segment towards the terminator. "Selective" culture conditions are those conditions that provide preferential growth of cells that have a predetermined phenotype. This phenotype is commonly the result of the expression of a gene that has been introduced into the cell (or introduced into a cell of origin) to complement a mutation. The "tandem duplication" of a chromosomal locus denotes the insertion into a chromosome of a second copy of an existing locus, whereby the second copy is inserted into the original copy by homologous recombination between the chromosomal locus and the counterpart locus in an exogenously supplied DNA molecule. Duplication may result in alteration of the original copy of the locus, such as when an altered form of the locus is introduced into the cell and incorporated into the chromosome. The resulting configuration of the duplicated locus is determined by the nature of any introduced alteration (s) and the presence or absence of additional DNA bound to the introduced locus copy. Within the methods of the present invention, tandem duplication and the target locus will generally introduce a disordered copy into the locus and insert a selectable marker or other vector sequences between the two locus copies. The present invention provides methods for introducing alterations (mutations) in the chromosomal loci of Pi chi a m e than ol i ca. Strains of Pi chi a me than ol i ca for use within the invention can be obtained from the American Type Culture Collection (Rockville, MD) and other depositories. These strains can be used as strains of origin for the production of strains having a desired chromosomal mutation. Those skilled in the art will recognize that both strains of origin and mutants can be further mutagenized according to known techniques in order to obtain strains having desired genotypes (eg, ade "). Thus, strains having definite nutritional requirements can be obtained. , metabolic defects, etc. In this way, it is possible to design strains of P. me olica for use, for example, in the large-scale fermentation of protein production, of particular interest for protein production systems. strains of P. me tha n ol i ca that are deficient in vacuolar protease activity In yeasts, the main storage of proteolytic activity is located within the vacuolar compartment lumen (Jones, Me th ods En zymol. 194: 428-453, 1991.) These proteases are released into the fermentation broth by spontaneous and inevitable cell lysis and are released further during the ru cellular ptura that is required to release proteins intracellularly produced in laboratory or industrial production, thus limiting the recovery of the intact protein. Therefore, it is desirable to reduce or eliminate the activity of the vacuolar protease in the production strains. Vacuolar protease genes of particular interest in this regard include the PEP4 gene encoding proteinase A, and the PRB1 gene, which codes for proteinase B. The designations of these genes are based on functional equivalence of the Saccharomyces cerevisiae genes. of the same names and by a high degree of sequence identity (70%) between the encoded proteins of P. methanolica and S. cerevisiae.

Although other vacuolar proteases (eg, carboxypeptidase Y) are present in P. methanolica, the gene products PEP4 and PRB1 activate the other vacuolar proteases, so that the negation of the functions of PEP4 and PRB1 results in a strain that is effective negatively to the vacuolar protease. The preparation of vacuolar protease deficient strains of P. methanolica as described herein serves to illustrate the methods of the present invention. Those skilled in the art will recognize that the described methods can be rapidly applied to the alteration of other chromosomal loci of P. methanolica. Other loci of interest in this regard include, without limitation, genes encoding alcohol oxidase (AUG1 and AUG2 genes), Golgi endoproteases (orthologs of the KEX2 and YAP3 genes of S. cerevisia e) nutritional markers (e.g. HIS3, LEU2), ß-1, 3-glucanase, and mating pheromones; the HO gene; and other genes encoding proteins of the methanol utilization pathway (eg, genes encoding dihydroxyacetone synthase, formate dehydrogenase and catalase). Within the present invention, an alteration is generated within a targeted, selected chromosomal locus through a connection / disconnection mutagenesis process, whereby an altered copy of the target chromosomal locus is used to replace the endogenous copy within of the genome. An example of this method is illustrated in Figure 1. A linear DNA construct comprising one (1) a portion of the target chromosomal locus selected in which at least one pair of nucleotides is altered (indicated by "?" In Figure 1), and (2) a selectable marker (ADE2 in Figure 1) in the cells. The cells are cultured under selective conditions,. then a subset of cells is identified, in which the portion of the altered chromosomal locus and the selectable marker has been integrated chromosomally by homologous recommendation. The recombination event results in tandem duplication of the target chromosomal locus. Within Figure 1, the introduced DNA is shown as a circular molecule, because P. I'm going to reform the linear DNA in a circle after the transformation. As shown in Figure 1, recombination between the introduced and chromosomal copies of the target locus results in the duplication of the target locus, with the selectable marker interposed between the two copies. The cells are then cultured under conditions in which cells suffering from a spontaneous disconnection event can be identified. This disconnection event results from a second homologous recombination between the two copies of the target locus. Two results are possible: the disconnection of the wild-type copy of the locus, illustrated in Figure 1; and the disconnection of the altered copy (not shown), which regenerates the primary state. An alteration is created at a cloned chromosomal locus or portion thereof, for example a cloned vacuolar protease gene, by conventional methods of DNA manipulation, such as by digestion with restriction endonucleases and insertion mutagenesis with re-ligation, reaction in polymerase chain (PCR; Mullis, U.S. Patent No. 4, 683, 202), sequence directed mutagenesis (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press , 1989, 15.3-15.113), or other methods known in the art. The altered copy of the locus is then introduced into the cell as a linear DNA construct that additionally comprises a selectable marker that complements an auxotrophic mutation in the cell. It is preferred that the cell be auxotrophic for adenine and that the selectable marker complements adenine auxotrophy. A preferred marker is the ADE2 gene of P. me tha n ol i ca, a representative sequence of which is shown in SEQ ID NO: 1, or a functional portion thereof. A population of P cells is prepared. m e than ol i ca, each one comprising a chromosomal copy of the target locus. The strains of P. I know they are available from publicly accessible depositories, such as the American Type Culture Collection, Rockville, MD, USA. The cells are cultured in a suitable medium, such as YEPD. -If necessary, the cells can be mutagenized to obtain the desired auxotrophy. To prepare auxotrophic mutants of P. me than ol i ca, the cells are first exposed to conditions of mutagenesis, that is, environmental conditions that cause genetic mutations in the cells. Methods for mutagenizing cells are well known in the art and include chemical treatment, exposure to ultraviolet use, exposure to X-rays, and insertional, retroviral mutagenesis. Chemical mutagens include ethylmethane sulfonate (EMS), N-met il-N'-nit ro-N-nit rosoguanidine, 2-methoxy-6-chloro-9- [3- (ethyl-2-chloroethyl) amino-propylamino] acridine «2HCL, -bromouracil, acridine, and aflatoxin. See, La rence, Methods Enzymol. 194: 273-281, 1991. The proportion of the mutilated cells obtained is a function of the concentration or amount of the mutagenizing agent to which the cells are exposed. A low level of mutagen produces a small proportion of mutant cells. High levels of mutagen produce a higher proportion of mutant cells, but also mutagenize more loci and kill more cells. Therefore, it is necessary to compensate for these results so that a reasonable number of slightly mutated cells is obtained. Compensation is generally made empirically by exposing cells to different conditions to establish a death curve. In general, the cells are exposed to mutagenesis conditions and are cultured for a day, after which they are tested for viability according to the normal assay methods. In general, it is preferred to use a level of mutagenesis that results in a 10-20% mortality, although one skilled in the art will recognize that this value can be adjusted, as necessary for example, if working with a very high number. large cell The mutagenized cells are then cultured in a rich medium to allow mutations to be established and replicated in at least a portion of the cell population. This step allows the cells in which the genome has been altered to replicate the mutation and pass it on to its progeny, thus establishing the mutation within the population. Then, the cells are transferred to a culture medium deficient in assimilable nitrogen so that the cellular storage of nitrogen is depleted. By "deficient in assimilable nitrogen" is meant that the medium lacks an amount of nitrogen sufficient to support the growth of the cells. The depletion of cellular nitrogen storage will generally require about 12 or 24 hours of incubation, with 16 hours being sufficient under common conditions. After depletion of the nitrogen stores, the cells are cultured in a defined culture medium comprising an inorganic nitrogen source and an amount of an antifungal antibiotic sufficient to stop the growth of the P cells. That's me. Antistatic nystatin (micos tat ina) is preferred. Preferred inorganic nitrogen sources are those that comprise ammonium ions such as ammonium sulfate. In general, the medium will contain 10-200 mM ammonium, preferably about 60 mM ammonium. Nystatin is included at a concentration of 0.1 to 100 mg / ml, preferably 0.5 to 20 mg / ml, more preferably about 2 mg / L (10 units / L). The treatment with nystatin is carried out for ten minutes to six hours, preferably about one hour. Those skilled in the art will recognize that the actual concentration of antibiotic and the exposure time required to kill the prototrophic cells can easily be determined empirically, and certain adjustments may be necessary to compensate for variations in specific activity between the individual batches of the antibiotic. By depleting cellular nitrogen stores and then culturing the cells in a defined medium containing an inorganic nitrogen and antibiotic source, cells that are auxotrophic for the amino acid or nucleotide biosynthesis remain alive because they can not grow in the cell. defined medium. The cells that grow are annihilated by the antibiotic. After antibiotic treatment, the cells are transferred to a rich culture medium. A significant proportion of the cells that survive nystatin treatment are prototrophic. The auxotrophic mutants within the surviving population they are identified and characterized when determining the nutrient requirements of the cells. Culture in replica plates is commonly used for this determination. The cells are plated in both rich medium and medium that lacks specific nutrients. Cells that do not grow on particular plates are auxotrophic for the absent nutrient. The complementation analysis can be used for additional characterization. Alteration of a chromosomal locus in the host cells is achieved by homologous recombination between the cell chromosomal locus and a segment of homologous DNA introduced into the cell. The homologous segment comprises at least a portion of the target locus that has been cloned into a DNA construct, typically a plasmid. At least one pair of nucleotides in the cloned portion of the locus is altered by its pressure, substitution and insertion, with the suppression being preferred. Combinations of the alterations can also be made, resulting, for example, in a cloned locus from which a first region and a second region that has been interrupted by insertion have been deleted. These alterations will preferably eliminate one or more amino acid residues from active sites of the protein product of the target locus, thereby distributing the activity of the protein. Mutations of structure change, for example, can be generated by deleting a partial codon, thereby suppressing a single nucleotide, and preferably at least four nucleotides, which can produce the desired inactivation mutation. It is preferred to suppress or otherwise alter at least the majority of the open reading frame (ORF) of the cloned locus. The alterations may extend beyond the ORF in the promoter or terminator or both, but it is preferred that they do not break the adjacent gene sequences. In practice, the actual extent of any deletion will usually be based on the locations of convenient restriction enzyme recognition sites. The alteration will be flanked at each end by a sufficient sequence to facilitate homologous recombination with the target locus. Although as little as two pairs of sequence identity base have been reported to be adequate (Mézard et al., Cell 10: 659-670, 1992), it is preferred to provide at least ten base pairs of the unaltered sequence of the locus. objective at each end of the alteration. It is preferred that the alteration be flanked by at least 100 base pairs, up to as much as 10 kilobase pairs, of the unaltered sequence at each end. In any case, the DNA construct must contain sufficient amounts of sequence homologous to the genome of the target cell to allow homologous recombination at the target locus. In practice, deletions or other alterations will commonly cover from about 1 kb to about 2 kb of the locus of interest, although larger regions of up to 10 kb or more may be altered depending on the size of the target locus. Within one embodiment of the invention, the object of the alteration is to effectively eliminate the activity of the target locus, and it is preferred to do so in a manner that will minimize or eliminate the possibility of setbacks or other compensation mutations. Within another modality, the object of the alteration is to insert a gene or genes that code for a new function. The application of the methods of the present invention to specific, objective loci is within the skill level of the art. As noted above, the DNA construct that is introduced into the cell will comprise, in addition to the altered locus portion, a selectable marker. The presence of the selectable marker facilitates the identification and selection of the integrating transformants by allowing cells expressing the marker to grow under conditions in which cells lacking the marker can not be multiplied. The general principles of selection are well known in the art. Commonly used selectable markers are genes that code for enzymes required for the synthesis of amino acids in nucleotides. Cells that have mutations in these genes can not grow on media lacking the specific amino acid or nucleotide unless the mutation complements the selectable marker. The use of these "selective" culture media ensures stable maintenance of the heterologous DNA within the host cell. A preferred selectable marker of this type, for use in Pi chi a me than ol i ca is an ADE2 gene from P. me thanol i ca, which codes for 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 sequence of the ADE2 gene of P. Representative representative is shown in SEQ ID NO: 1. The illustrated sequence includes 1006 nucleotides of the 5 'non-coding sequence and 442 nucleotides of the 3' non-coding sequence, with the ATG initiation codon in the 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 may be used while the coding portion is operably linked to the promoter and terminator sequences. Those skilled in the art will recognize that this and other sequences provided herein represent individual alleles of the respective genes, and that allelic variation is expected. Any functional ADE2 allele can be used as a selectable marker. Other alternative nut markers that can be used within the present invention include the genes ADE1, HIS3 and LEU2 of P. me than ol i ca, which allow selection in the absence of adenine, histidine and leucine, respectively. Clones of P. m e than ol i ca can be cloned based on homology with their genes from Saccharomyces cerevisiae to their counterparts. Heterologous genes, such as the genes of other fungi, are also. They can use as selectable markers.

The DNA that is introduced in P. I know that ICA is linearized first, such as by digestion with one or more restriction endonucleases. Linearization increases transformation efficiency. The linearization by digestion with endonuclease is sequence specific also allows the specific removal of the exogenous sequences, so that only the desired DNA sequences are introduced into the cells. DNA can be introduced into P cells. I thank you for any of the several known methods, including lithium transformation (Hiep et al., Yeast 9_: 1189-1197, 1993; Tarutina and Tolstorukov, Abst. Of the 15th International Specialized Symposium on Yeasts, Riga (USSR ), 1991, 137, Ito et al., J. Bacteriol., 153: 163, 1983, Bogdanova et al., Yeast 1: 1, 343, 1995), transformation by air bubbles (Beggs, Nature 275: 104, 1978; et al., Proc. Nati. Acad. Sci. USA 7_5_: 1929, 1978; Cregg et al., Mol. Cell. Biol. 5_: 3376, 1985), transformation by polyethylene glycol with freeze-thaw (Pichia Expressión Kit Instruction Manual, Invitrogen Corp., San Diego CA, Cat. No. K1710-01), or elect roctoration, the latter being preferred. Electing is the process of using an electric impulse field to transiently permeate cell membranes, allowing macromolecules, such as DNA, to pass into cells. Electroporation has been described for use with mammalian host cells (e.g., Neuman et al., EMBO J. 1 ^: 841-845, 1982) and fungal (e.g., Meilhoc et al., Bio / Technology 8_: 223-227, 1990). However, the actual mechanism by which DNA is transferred to cells is not well understood. For the transformation of P. me than ol i ca, it has been found that electroporation is surprisingly efficient when the cells are exposed to an exponentially decaying electric field that have a field strength of 2.5 to 4.5 kv / cm and a time constant (t) from 1 to 40 milliseconds. The time constant (t) is defined as the time required for the initial peak voltage V0 that falls to a Vo / e value. The time constant can be calculated as the product of the total resistance and the pulse circuit capacitance, ie, (t) = R x C. Typically, the resistance and capacitance are either pre-adjusted or can be selected by the user, depending on the electroporation equipment selected. In any case, the equipment is configured in accordance with the manufacturer's instructions to provide a field strength and decay parameters as described above. Electroporation equipment is available from commercial suppliers (eg, BioRad Laboratories, Hercules, CA). Cells into which the altered locus has been introduced are then cultured under conditions that are selective for the presence of the selectable marker as described above. The cells obtained after culturing under selective conditions are then analyzed to identify a subset of the cells in which the altered locus and the selectable marker by chromosomal integration have been chromosomally integrated by homologous recombination. Tandem duplication of the target chromosomal locus resulting from homologous recombination can be detected by structural changes at the target locus. Transformants that have undergone the desired homologous recombination event are identified by known methods, such as Southern blotting (see, for example, Strathern and Higgins, Me th o.ds In zymol., 194: 319-329, 1991) or the reaction in polymerase chain. For Southern blotting, genomic DNA is prepared from the transformants and control cells, digested with one or more restriction enzymes, transferred to a blot, and probed to detect a change in the restriction pattern after the transformation. The reagents, materials, equipment and protocols to prepare and probe transfers are available from commercial suppliers. In the alternative, the target region can be amplified by PCR and analyzed by gel electrophoresis to detect a change in size. As described in more detail in Example 3, below, the selection for the Ade + phenotype gives rise to two kinds of transformants. A class originates rapidly as white colonies on the primary translation plate. The second class becomes evident as fast-growing white papids at the edges of the unstable, pink transformants. Southern blot analysis showed that 19% of the first class of transformants had undergone homologous recombination, while 89% of the cells of the white porridges were homologous recombinants. Cells that have tandem duplication, then desired, are cultured under conditions where the prototrophic cells grow and exhibit a first phenotype, and the auxotrophic cells grow and exhibit a second phenotype. The culture conditions are non-selective to allow the spontaneous disconnection of the selectable, integrated marker and the wild-type copy of the target locus as shown in Figure 1. This phenotypic differentiation is achieved for example by culturing the cells in rich medium containing amounts of adenine binding, so that cells can grow ade "but colonies ade" are pink. The auxotrophic cells (ade ") are then recovered.A subset of the auxotrophic cells in which the altered locus has been chromosomally integrated, is then identified using conventional analytical methods, such as PCR or restriction enzyme digestion and Southern blotting as described earlier Mitotic recombination may result in the disconnection of any copy of the duplicated locus in tandem.The desired cells are those in which the selectable marker and the wild type chromosomal sequences have been disconnected, leaving a disordered, individual copy of the Chromosomal locus, target (Figure 1) The presence of an alteration in the target chromosomal locus can be further confirmed by assays, including activity assays, endonuclease digestion and Southern blot analysis, and growth phenotype assays. In certain cases, it may be necessary to alter a lurality of the locus in order to obtain the desired phenotype. For example, vacuolar protease deficiency is obtained by eliminating the activities of proteinase A and proteinase B. Vacuolar protease activity (and therefore vacuolar protease deficiency) is measured using any of several known assays. Preferred assays are those developed for Saccharomyces cervi s i a e and described by Jones, Methods Enzymol. 194: 428-453, 1991. A preferred test of this kind is the APE coating assay, which detects the activity of carboxypeptidase Y (CpY). Briefly, the assay detects the release of β-naphthol mediated by carboxypeptidase Y from an ester, which results in the formation of an insoluble red dye by the reaction of β-naphthol with the diazonium salt Fast Garnet GBC. Colonies are coated with a 0.6% agar solution of the N-acetyl-DL-phenylalanine β-naphthyl ester containing 1 mg / ml dimethylformamide. After the coating hardens, the plates are flooded with a solution of Fast Garnet GBC (5 mg / ml in 0.1 M Tris-HCL, pH 7.3-7.5). Within a few minutes, the Cpy + colonies turn red. The activity of carboxypeptidase Y can also be detected by the well test in which the cells are distributed in wells of a microtiter test plate and iated in the presence of N-benzoyl-L-tyrosine-p-nitroanilide (BTPNA ) and dimethylformamide. Cells are permeabilized by dimethylformamide, and CpY in cells sinde amide bound in BTPNA to give the yellow product, p-ni t roanilina. Assays for CpY will detect any mutation that reduces the protease activity while the activity ultimately results in the reduction of CpY activity. Proteinase B activity can be detected using an HPA coating test, which detects the solubilization of Azur Skin Powder by proteinase B. Enzyme-producing colonies are circled by a clear halo, while deficient mutants remain uncovered. Carboxypeptidase S can be assessed using a well test that detects the release of leucine from carbobenzoxyglycyl-L-leucine. In the presence of L-amino-acid-oxidase, H202 is produced by the oxidation of free leucine. H202 reacts with o-dianisidine dihydrochloride in the presence of peroxidase to produce oxidized dianisidine, which is dark brown. Additional assays are known and are within the skill level in the art to be performed. The strains that have the target, altered loci are useful as hosts for the expression of heterologous genes. Methods for introducing heterologous DNA into P. I believe that to cultivate the cells and to express the heterologous genes are described in WIPO Publication WO 9717450. The cells that are to be transformed with the heterologous DNA will have a mutation that can be complemented by a selectable marker in the molecule of heterologous DNA. Because the selectable marker is cleaved from the chromosome in the disconnection step described above, it is convenient to use the same marker in the introduction of a gene encoding the protein of interest. However, those skilled in the art will recognize that a different marker can be used. The selection of particular cell and marker combinations is within the skill of experience in the art. The proteins that can be produced in P. me than ol i ca include proteins of industrial interest and pharmaceutical. These proteins include higher eukaryotic proteins of plants and animals, particularly vertebrate animals such as mammals, although certain proteins are also of great value from microorganisms. Examples of protein that can be prepared 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 ina; and hormones such as insulin, leptin and glucagon. DNA molecules for use in the transformation of P. me than ol i ca are commonly prepared as circular double-stranded plasmids, which are preferably linearized before transformation. For protein or polypeptide production, the DNA molecules will include, in addition to the selectable marker described above, an expression cartridge comprising a transcription promoter, a DNA segment (e.g., a cDNA) that encodes the polypeptide or protein of the invention. interest, and a transcription terminator. These elements are operably linked to provide the transcription of the DNA segment of interest. It is preferred that the promoter and terminator be those of a P gene. m e than ol i ca. Useful promoters include those of constitutive and methanol-inducible promoters. The promoter sequences are generally contained in the space of 1.5 kb in the 5 'direction of the coding sequence of a gene, often in the space of 1 kb or less. In general, the regulated promoters are greater than the constitutive promoters due to the presence of regulatory elements. The methanol-inducible promoters, which include both positive and negative regulatory elements, may extend to more than 1 kb in the 5 'direction of the initiation ATG. The promoters are identified by function and can be cloned according to known methods. A particularly preferred methanol inducible promoter is that of a P alcohol utilization gene. me than ol i ca. A coding strand sequence, representative of this gene, AUG1 is shown in SEQ ID NO: 2. Within SEQ ID NO: 2, the initiation ATG codon is at nucleotides 1355-1357. Nucleotides 1-23 of SEQ ID NO: 2 are non-AUG1 polylinker sequences. Particularly it is preferred to use as a promoter a segment comprising nucleotides 24-1354 of SEQ ID NO: 2, although an additional sequence 5 'may be included. The P. me than ol i ca contains a second alcohol utilization gene, in AUG2, the promoter of which can be used within the present invention. In SEQ ID NO: 3 a partial DNA sequence of an AUG2 clone is shown. The segments of the AUG2 promoter used during the present invention will generally comprise nucleotides 91-169 of SEQ ID NO: 3, although small truncations at the 3 'end will not be expected to cancel the function of the promoter. Other useful promoters include those of dihydroxyacetone-sint asa (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. Genes coding for these enzymes from other species have been described, and their sequences are available (eg Jano icz et al., Nuc Acids Res. 13_: 2043, 1985, Hollenberg and Janowicz, EPO publication 0 299 108; Didion and Roggenkamp, FEBS Lett 303: 113, 1992). The genes encoding these proteins can be cloned by using the sequences known as probes, or by aligning known sequences, designing values based on the alignment, and amplifying the P-DNA. I like it because of the polymerase chain reaction. Constitutive promoters are those that are not activated or inactivated by environmental conditions; they are always transcriptionally active. Preferred constitutive promoters for use within the present invention include those of glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, and phosphoglycerate kinase genes of P. That's me. These genes can be cloned as described previously or by complementation in a host cell, such as a Saccharomyces cerevisiae cell, which has a mutation in the counterpart gene. Mutants of this type are well known in the art. See, for example, Kawasaki and Fraenkel, Biochem. Biophys. Res. Comm. 108: 1107-1112, 1982; McKnight et al., Cell 4_6: 143-147, 1986; Aguilera and Zimmermann, Mol. Gen. Genet. 202: 83-89, 1986. The DNA constructs used within the present invention may additionally contain additional elements, such as an origin of replication and a selectable marker that allows an amplification and maintenance of the DNA in an alternate host (e.g. E. col i). 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 the selectable marker, flanked at both ends by the host DNA sequences. This is conveniently achieved by including the 3 'untranslated DNA sequence at the end in the 3' direction of the expression segment and depending on the promoter sequence at the 5 'end. When linear DNA is used, the expression segment will be flanked by cleavage sites to allow linearization of the molecule and separation of the expression segment from other sequences (eg, a bacterial origin of replication and selectable marker). Preferred are cleavage sites that are recognized by restriction endonucleases that cut infrequently within a DNA sequence, such as those that recognize the target sequences of 8 bases (e.g., Not I). The heterologous DNA is introduced into P. me than ol i ca cells as described above. Electroporation is a preferred method. The Electroporation of P. me than ol i ca is carried out preferably in cells in early phase growth. The cells typically become electrocompetent by incubating them at about 30 ° C for about 5 to 30 minutes in a buffered solution of pH 6-8 containing a reducing agent, such as dithiothreitol (DTT) or β-mercaptoethanol (BME), to reduce the cell wall proteins, to facilitate the subsequent admission of DNA. The cells are then harvested and washed in a suitable electroporation buffer, which is used cooled in ice. Suitable buffers in this regard include solutions of pH 6-8 containing a weak buffer, divalent cations (e.g., Mg ++, Ca ++) and an osmotic stabilizer (e.g., a sugar). A preferred electroporation buffer is STM (270 mM sucrose, 10 mM Tris, pH 7.5, 1 mM MgCl 2). Within a preferred protocol, cells are subjected to two washes, first in the original culture volume of the ice-cooled buffer, and then in half of the original volume. After the second wash, the cells are harvested and re-dispersed, typically using about 3-5 ml of buffer for an original 200 ml culture volume. Electroporation is carried out using a small volume of elect rocompetent cells (typically about 100 μl) and up to one tenth volume of linear DNA molecules. For example, 0.1 ml of cell suspension is combined in a buffer that does not exceed 50 mM in ionic concentration, with 0.1-10 mg of DNA (vol <10 ml). This mixture is placed in an ice-cooled electroporation probe and subjected to an electric pulse field from 2.5 to 4.5 kV / cm, preferably close to 3.75 kV / cm, and a time constant of approximately 20 milliseconds, with exponential decay . After they have been treated with pulses, the cells are diluted approximately 10X in 1 ml of YEPD broth and incubated at 30 ° C for one hour. The cells are then harvested and plated on selective media. Cells having the ade2 mutations that have been transformed with the selectable ADE2 material can be plated in minimal medium lacking adenine, such as ADE D (Table 1) or ADE DS (Table 1). In a typical procedure, 250 ml aliquots of cells are plated on 4 separate ADE D or ADE DS plates to select Ade + cells. For the production of proteins, the cells of P. me than ol i ca are grown in a medium that includes 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 a conventional means, such as agitation of small flakes or dew of the burners. A preferred culture medium is YEPD (Table 1). The cells can be passed by dilution in fresh culture medium or stored for short periods in plates under refrigeration. For long-term storage the cells are preferably kept in a glycerol solution at 50% -70 ° C.

Table 1 YEPD 2% D-Glucose 2% Peptone Bacto ™ (Difco Laboratories, Detroit, MI) 1% yeast extract Bacto ™ (Difco Laboratories) 0.004% adenine 0.006% L-leucine ADE D 0.056% powder - Ade -Trp -Thr 0.67% yeast nitrogen base without amino acids 2% D-glucose 0.5% tryptophan at 200X, threonine solution ADE DS 0.056% powder -Ade -Trp -Thr 0.67% nitrogen base yeast without amino acids 2% of D-glucose 0.5% of tryptophan at 200X, solution of trianine 18.22% of D-sorbitol LEU D 0.52% of powder of -Leu -Trp -Thr 0.67% of base of yeast nitrogen without amino acids 2% of D-glucose 0.5% tryptophan 200X, threonine solution HIS D 0.52% -His -Trp -Thr powder 0.67% of yeast nitrogen base without amino acids 2% of D-glucose 0.5% of tryptophan 200X, solution of Threonine URA D 0.56% Powder of -Ura -Trp -Thr 0.67% of base of yeast nitrogen without amino acids 2% of D-glucose 0.5% of tryptophan 200X, Threonine solution URA DS 0.56% powder -Ura -Trp -Thr 0.67% yeast nitrogen base without amino acids 2% D-glucose 0.5% tryptophan 200X, threonine solution 18.22% D-sorbitol Dust - Leu -Trp -Thr powder made by combining 4.0 g of adenine, 3.0 g of arginine, 5.0 g of aspartic acid, 2.0 g of histidine, 6.0 g of isoleucine, 4.0 g of plant, 2.0 g of methionine, 6.0 g of phenylalanine, 5.0 g of serine, 5.0 g of tyrosine, 4.0 g of uracil and 6.0 g of valine (all L-amino acids) Powder of -His -Trp -Thr powder made by combining 4.0 g of adenine, 3.0 g of arginine, 5.0 g of aspartic acid, 6.0 g of isoleucine, 8.0 g of leucine, 4.0 g of plant, 2.0 g of methionine, 6.0 g of phenylalanine, 5.0 g of serine, 5.0 g of tyrosine, 4.0 g of uracil and 6.0 g of valine (all L - amino acids) -Ura -Trp -Thr powder made by combining 4.0 g of adenine, 3.0 g of arginine, 5.0 g of aspartic acid, 2.0 g of histidine, 6.0 g of isoleucine, 8.0 g of leuci na, 4.0 g of lysine, 2.0 g of methionine, 6.0 g of phenylalanine, 5.0 of serine, 5.0 g of tyrosine and 6.0 g of valine (all L-amino acids) Powder of -Ade -Trp -Thr powder made by combining 3.0 g of arginine, 5.0 g of aspartic acid, 2.0 g of histidine, 6.0 g of isoleucine, 8.0 g of leucine, 4.0 g of lysine, 2.0 g of methionine, 6.0 g of phenylalanine, 5.0 g of serine, 5.0 g of tyrosine, 4.0 g of uracil and 6.0 g of valine (all L-amino acids) Triptofan 200X, threonine solution 3.0% L-threonine, 0.8% L-tryptophan in H20. For plates, add 1.8% BactoMR agar (Difco Laboratories) The P. It recognizes that certain frequencies that occur infrequently, called autonomous replication sequences (ARS), as origins of DNA replication, and these sequences may occur fortuitously within a DNA molecule used for transformation, allowing that the transformation DNA remains ext-chromosomal. However, integrating transformants are generally preferred for use in the protein production system. These cells can be propagated without continuous selective pressure because the DNA is only lost in the genome. The integration of the DNA into the host chromosome can be confirmed by analysis with Southern blots. Briefly, the transformed and untransformed host DNA is digested with endonucleases. of restriction, is separated by electrophoresis, transferred to a supporting membrane, and probed with appropriate DNA host segments. The differences in fragment patterns seen in untransformed and transformed cells are indicative of integrative transformation. Restriction enzymes and probes can be selected to identify the segments of the transforming DNA (e.g., promoter, terminator, heterologous DNA, and selectable marker sequences) from among the genomic fragments. Differences in the expression levels of the heterologous proteins can result from factors such as the integration site and the copy number of the expression cartridge and the differences in promoter activity between the individual isolates. Thus, it is advantageous to detect or select a number of asylees for the level of expression before selecting a production strain. A variety of suitable detection methods are available. For example, transformant colonies are grown on plates that are coated with membranes (eg, nitrocellulose) that bind to the protein. The proteins are released from the cells by secretion or after lysis, and bind to the membrane. The bound protein can then be assessed using known methods, including immunoassays. More accurate analyzes of expression levels can be obtained by culturing cells in a liquid medium and analyzing the conditioned medium or cellular media, as appropriate. The methods for concentrating and purifying proteins from the media used will be determined in part by the protein of interest. These methods are easily selected and practiced by one skilled in the art. For small-scale protein production (e.g., shake flask or plate production) the P. me than ol i ca transformants having an expression cartridge comprising a methanol-regulated promoter (such as the AUG1 promoter) are they cultivate in the presence of methanol and the absence of interference amounts from other carbon sources (eg, glucose). For small-scale experiments, including preliminary detection of expression levels, transformants can be cultured at 30 ° C in solid media containing, for example, 20 g / L of Bacto-agar (Difco), 6.7 g / L of base of yeast nitrogen without amino acids (Difco), 10 g / L of methanol, 0.4 mg / L of biotin and 0.56 g / L of powder -Ade -Thr -Trp. Because methanol is a volatile source of carbon, it is easily lost in prolonged incubation. A continuous supply of methanol can be provided by placing a 50% methanol solution in water in the caps of the inverted plates, whereby the methanol is transferred to the growing cells by evaporative transfer. In general, no more than 1 mL of methanol per 100 mm plate is used. Experiments on a slightly larger scale can be carried out using cultures grown in shake flasks. In a typical procedure, the cells are cultured for two days in minimal methanol plates as described above at 30 ° C, then the colonies are used to inoculate a small volume of minimal methanol medium (6.7 g / L base). yeast nitrogen without amino acids, 10 g / L methanol, 0.4 mg / L biotin) at a cell density of approximately 1 x 106 cells / ml. The cells are cultured at 30 ° C. Cells growing in methanol have a high oxygen requirement, requiring vigorous agitation during culture. The methanol is replenished daily (typically 1/100 volumes of 50% methanol per day). For cultivation on a production scale, fresh cultures of high production clones are prepared in shake flasks. The resulting cultures are then used to inoculate the non-fermented culture medium. Typically, a 500 ml culture in YEPD grown at 30 ° C for 1-2 days with vigorous shaking is used to inoculate a 5 liter fermenter. The cells are cultured in a suitable medium containing salts, glucose, biotin, and trase elements at 28 ° C, pH 5.0, and 02 dissolved at >30% After the initial glucose load is consumed (as indicated by a decrease in oxygen consumption), a glucose / methanol feed is distributed to the container to induce production of the protein of interest. Because large scale fermentation is carried out under carbon feeding conditions, the presence of glucose in the feed does not repress the methanol inducible promoter. The use of glucose in combination with methanol under limited glucose conditions produces rapid growth, efficient conversion of carbon to biomass and rapid changes in physiological growth states, while still providing complete induction of methanol-inducible gene promoters. In a typical run of fermentation, a cell density of about 80 to about 400 grams of wet cell paste per liter is obtained. "Wet cell paste" refers to the mass of cells obtained by harvesting the cells from the fermenter, typically by culture centrifugation. For industrial processes, on a large scale, where it is desirable to minimize the use of methanol, it is preferred to use host cells that have a genetic defect in a gene required for the use of methanol. These genes include the AUG1 and AUG2 genes of alcohol oxidase as well as the genes encoding catalase, formaldehyde dehydrogenase, formate-dehydrogenase, dihydroxyacetone-synthase, dihydroxyacetone-kinase, fructose, 1,6-bis-fos-o-aldolase, and Fructose 1,6-bisphosphatase. It is particularly preferred to use cells in which both alcohol oxidase genes (AUG1 and AUG2) are deleted or altered in order to eliminate their activity. This alteration can be achieved by the on / off method of the present invention or the selected gene replacement. The invention is further illustrated by the following non-limiting examples EXAMPLES Example 1 The cells of P. me tha n ol i ca (strain CBS6515 of American Type Culture Collection, Rockville, MD) were mutagenized by UV exposure. A death curve was generated first by plating cells on 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 a period of time as shown in table 2. The plates are then protected from visible light sources and incubated at 30 ° C for two days.

Table 2. Viable cells Plate Time 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 L 6 sec. 0 2 1 Then a large-scale mutagenesis was carried out using a 2-second UV exposure to provide 20% death. Cells were plated at approximately 10 4 cells / plate in 8 YEPD plates that were subcomplemented with 100 mg / L each of uracil, adenine, and leucine, which were added to complement the growth of potential auxotrophs having inbred deficiencies . After exposure to UV, the plates were rolled into sheets and incubated overnight at 30 ° C. The next day, the colonies on the plates (~ 105 total) were re-dispersed in water and washed once with water. A sufficient amount of cell suspension was used to give an OD600 of 0.1 - 0.2 to inoculate 500 ml of minimal broth made with yeast nitrogen base without amino acids or ammonia, supplemented with 1% glucose and 400 μg / L biotin. The culture was plated in a Bell flask with 2.8 liter defectors and stirred vigorously overnight at 30 ° C. The next day the cells have reached an OD6oo of about 1.0 - 2.0. The cells were pelleted and re-dispersed in 500 ml of minimal broth supplemented with 5 g / 1 of ammonium sulfate. The cell suspension was plated in a Bell flask with 2.8 L baffles and shaken vigorously at 30 ° C for 6 hours. 50 ml of culture were removed in a 250 ml flask with a control, and 1 mg of nystatin (Sigma Chemical Co., St. Louis, MO) was added to the rest of the culture to select the 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 re-dispersed at OD60o of 1.0 in 50% glycerol and frozen. Titration of cells treated with nystatin against control cells for colony forming units revealed growth with nystatin decreased the number of viable cells by a factor of 104"10 ~ 2 dilutions of cells treated with nystatin were plated in 15 YEPD plates, the colonies were plated on replicate plates in minimal plates (2% agar, 1 x YNB, 2% glucose, 400 μg / L biotin), the frequency of the auxotrophs was approximately 2 - 4. % Approximately 180 auxotrophic colonies were collected on YEPD + Ade, Leu, Ura plates and plated with replicate to several marginal plates.All auxotrophs were Ade ". Of these, 30 were remarkably pink on marginal plates (LEU D, HIS D, etc., see Table 1). Of the 30 pink mutants, 21 were chosen for further study; the rest was either weak to grow on AD D plates or were contaminated with wild-type cells. The Ade mutants were then subjected to complementation analysis and phenotypic testing.To determine the locus number defined by mutants, the 21 mutants were mated to a "pink" Ade strain (strain # 2). Mating is carried out by mixing cell suspensions (OD6oo = 1) and plating the mixtures in 10 μl aliquots on YEPD plates). The cells were then replicated to SPOR medium (0.5% Na-acetate), 1% KCl, 1% glucose, 1% agar) and incubated overnight at 30 ° C. The cells were then plated with ADE D replica plate for phenotype classification. As shown in Table 3, some combinations of mutants failed to give Ade + colonies (possibly defining the same genetic locus as in strain # 2), while others give rise to numerous Ade + colonies (possibly defining a separate genetic locus). ). Because mutant # 3 gave Ade + colonies when paired with # 2, the complementation test with mutant # 3 was repeated. If the mutant group defined two genetic loci, then all mutants failed to give Ade + colonies. mated to strain # 2 should give Ade + colonies when mated to # 3. The results of the crosses are shown in Table 3.

Table 3 ufantes x Mutant # 2 x Mutant # 3 # 1 + # 3 + # 10 + - # 15 + # 18 + # 24 + # 28 + # 30 + # 2 - + # 6 - + # 8 - + # 9 - + # 11 - + # 17 - + # 19 - + # 20 - + # 22 - + # 27 - + # 4 + + # 12 + + # 16 + + As shown in Table 3, most of the mutants fall into one of two groups, consistent with the The idea is that there are two adenosine biosynthetic genes that, when absent, result in pink colonies in limiting adenine media. Three colonies (# 4, # 12, and # 16) can define either a third locus or exhibit intragenic complementation. Two intensely pigmented mutants from each of the two complementation groups (# 3 and # 10; # 6 and # 11) were selected for further characterization. Further analysis indicated that Ade "was the only auxotrophy present in these strains.A bank of clones of P. me olica in the vector pRS426, a transporter vector comprising 2μ and sequences URA3, of S. cerevi si, was constructed. ae, allowing them to propagate in S. cerevi if ae. Genomic DNA was prepared from strain CBD6515 according to normal procedures.Briefly, the cells were grown overnight in rich medium, spheronated with zimolyase, and The DNA was precipitated in the lysate with ethanol and extracted with a mixture of phenol / chloroform, and then precipitated with ammonium acetate and ethanol.The gel electrophoresis of the DNA preparation showed the presence of the high molecular weight, intact, and appreciable amounts of RNA.The DNA was then partially digested with Sau 3A by incubating the DNA in the presence of a serial dilution of the enzyme. r electrophoresis to determine the size distribution of the fragments. The DNA that migrates between 4 and 12 kb was cut from the gel and extracted from the gel cut. The DNA fractionated in size was then ligated to PRS426 which has been digested with Bam Hl and treated with alkaline phosphatase. Aliquots of the reaction mixture were electroporated into MC1061 E cells. Col i using the BioRad Gene Pulser RM device as recommended by the manufacturer. The genomic library was used to transform the HBY21A strain of S. cerevi s i a e (ade2 ura3) by electroporation (Becker and Guarente, Methods Enzymol 194: 182-187, 1991). The cells were re-dispersed in 1.2 M sorbitol, and 300 aliquots of 300 μl were plated on ADE D, ADE DS, URA D and URA DS plates (Table 1). The plates were incubated at 30 ° C for 4-5 days. Ade + colonies were not recovered on the ADE D or ADE DS plates. The colonies of the URA D and URA DS plates were plated with replica to the ADE D plates and two closely spaced white colonies were obtained. These colonies were re-veined and confirmed to be Ura + and Ade + 'These two strains, designated Adel and Ade6, were veined in the medium 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 "in replicate plating These results indicate that Ade + complementation activity is genetically linked to the URA3 marker carried by plasmid Plasmids obtained from yeast strains Adel and Ade6 appeared to be identical by restriction co-relation as described below.These genomic clones were designated pADEl-1 and pADEl-6, respectively.The total DNA was isolated from the Adel and Ade6 transformants of HBY21A and used to transform Strain MC1061 from E. col. Amp.RTM. DNA was prepared from two Adel AmpR colonies and 3 Amp6 colonies from Ade6. DNA was run with Pst I, Sea I, and Pst I + Sea I and analyzed by elect roforesis gel The five isolates produced the same restriction pattern.

The PCR primers were designed from the published sequence of the ADE2 gene of P. me than ol i ca (also known as ADE1; Hiep et al., Yeast 9_: 1251-1258, 1993). Primer ZC9080 (SEQ ID NO: 4) was designed to be primed on bases 406-429 of DNA of ADE2 (SEQ ID NO: 1), and primer ZC9079 (SEQ ID NO: 5) was designed to prime on the bases 2852-2829. Both primers included the limbs to introduce the 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 the plasmid DNA of the five putative ADE2 clones as the template DNA. The 100 ml reaction mixtures contained lx Taq PCR buffer (Boehringer Mannheim, Indianapolis, IN) 10-100 ng of plasmid DNA dNTPs, 0.25 mM, 100 pmol of each primer and 1 μl of Taq polymerase (Boehringer Mannheim). The 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 produced a PCR product of the expected size (2.4 kb). The co-relation by restriction of the DNA fragment of a reaction gave the fragments of expected size when digested with Bgl II or Sal I. The positive PCR reactions were mixed and digested with Spe I. The vector pRS426 was digested with Spe I it was treated with calf intestine phosphatase. 4μl of the PCR fragment and 1μl of the vector DNA were combined in a lOμl reaction mixture using standard ligation conditions. The ligated DNA was analyzed by gel electrophoresis. Digestions with Spe I were analyzed to identify plasmids that have a subclone of the ADE2 gene within pRS426. The correct plasmid was designated pCZRlld. Because the ADE2 gene in pCZRlld has been amplified by PCR, it is possible that mutations have been generated that are disabled from the functional character of the gene. To test these mutations, the subclones with the insert of interest were transformed individually into strain HBY21A of Sa ccha romyces cerevi s i a e. The cells were made electrocompetent and transformed according to normal procedures. They were placed on transformant plates in URA D and ADE D plates. Three phenotypic groups were identified. Clones 1, 2, 11, and 12 gave strong growth of many transformants in ADE D. The transformation frequency was comparable to the frequency of the Ura + transformants. Clones 6, 8, 10 and 14 also gave a high transformation efficiency to both Ura + and Ade +, but. Ade + colonies were somewhat smaller than those in the first group. Clone 3 gave many Ura + colonies, but no Ade + colonies, suggesting that it has a non-functional de2 mutation. Clones 1, 2, 11, and 12 were mixed. To identify the complementation group at de2 of P. m e than ol i ca, two representative mutants of each complementation group (# 3 and # 10 were transformed with the cloned ADE gene); # 6 and # 11), which were selected based on deep red pigmentation when they grow in limiting adenine. Two 100 ml cultures of the early phase cells were harvested by centrifugation at 3000 xg for 3 minutes and re-dispersed in 20 ml of fresh KD buffer (50 mM potassium phosphate buffer, pH 7.5, containing DTT 25). mM). The cells were incubated in this buffer at 30 ° C for 15 minutes. The cells were then harvested and re-dispersed in 200 ml of ice cold STM (sucrose at 270 mM, 10 mM Tris, pH 7.5, 1 mM MgCl2). The cells were harvested and re-dispersed in 100 ml of ice cold STM. The cells were harvested again and re-dispersed in 3-5 ml of ice cold STM. Aliquots of 100 μl of electrocompetent cells from each culture were then mixed with pADEl-I DNA. The cell / DNA mixture was placed in a 2 mm electroporation tube and subjected to a pulse electric field of 5kV / cm using a BioRad Gene PulserRM adjusted to a resistance of 1000O and capacitance of 25 μF. After being treated with pulses, the cells were diluted by the addition of 1 ml YEPD and incubated at 30 ° C for one hour. The cells were then harvested by gentle centrifugation and re-dispersed in 400 μl of minimal selective medium lacking adenine (ADE D). The re-dispersed samples were diluted in 200 μl aliquots and placed on ADE D and ADE DS plates. The plates were incubated at 30 ° C for 4-5 days. Mutants # 6 and # 11 gave the Ade + transformants. No Ade + transformants were observed when the DNA was omitted, therefore the two isolates seemed to define the de2 complementation group. The sequence of ADE2 is shown in SEQ ID NO: 1.

Example 2 The clone bank of P. methanolica discussed in Example 1 was used as a source for cloning the alcohol utilization gene (AUG1). The clone bank was stored in separate mixtures, each representing about 200-250 individual genomic clones. 0.1 ml of the "miniprep" DNA in each mixture was used as a template in the polymerase chain reaction with PCR primers (ZC8784, SEQ ID NO: 6, ZC8787, SEQ ID NO: 7) that were designed from an alignment of sequences conserved in alcohol genes with oxidase from Hansenula polymorpha, Candida boidini, and Pichia pastor is. The amplification reaction was run for 30 cycles of 94 ° C, seconds; 50 ° C, 30 seconds; 72 ° C, 60 seconds; followed by a 7 minute incubation at 72 ° C. A mixture (# 5) gave a band of approximately 600 bp (Figure 2). DNA sequencing of this PCR product revealed that it encoded an amino acid sequence with a sequence identity of approximately 70% with alcohol oxidase from Pichia pastor is encoded by the OAX1 gene and approximately 85% sequence identity with alcohol -oxidase of Hansenula polymorpha encoded by the M0X1 gene. The sequence of the AUG1 gene is shown in SEQ ID NO: 2. The sub-mixtures of the # 5 mixture were analyzed by PCR using the same primers used in the initial amplification. A positive sub-mixture was further separated to identify a positive colony. This positive colony was plated, and the DNA was prepared from individual colonies. Three colonies gave identical patterns after digestion with Co. I. The restriction correlation of the genomic clone and the PCR product revealed that the AUG1 gene is in a 7.5 kb genomic insert and that the sites within the PCR set could be explained only inside the genomic insert (Figure 2). Because the orientation of the gene within the PCR fragment is known, this latter information provided the approximate location and direction of transcription of the AUG1 gene within the genomic insert. DNA sequencing within this region revealed that with a very high sequence similarity at the amino acid level to other known alcohol-oxidase genes.

Example 3 To generate a strain of P. methanolica deficient for vacuolar proteases, the PEP4 and PRB1 genes were identified and broken. The sequence of PEP4 and PRB1 were amplified by PCR in a reaction mixture containing 100 pmol of primer DNA, buffer IX as supplied (Boehringer Mannheim, Indianapolis, IN) 250 mM dNTPs, 1-100 pmol of template DNA, and a unit of Taq-polymerase in a reaction volume of 100 ml. The DNA was amplified for 30 cycles of 94 ° C, 30 seconds, 50 ° C, 60 seconds; and 72 ° C, 60 seconds. The use of a sequence alignment of PEP4 was derived from S. cerevisiae (Ammerer et al., Mol Cell. Biol. 5: 2490-2499, 1986; Woolford et al., Mol.

Cell. Biol. 6: 2500-2510, 1986) and P. pastoris (Gleeson et al., Patent of the United States Number 5,324,660), several homosense and antisense primers were designed corresponding to the conserved regions. A set of primers, ZC9118 (SEQ ID NO: 8) and ZC9464 (SEQ ID NO: 9) produced a PCR product of the expected size from the genomic DNA and this set was used to identify a genomic clone corresponding to the amplified region. DNA sequencing of a portion of this genomic clone (shown in SEQ ID NO: 10) revealed an open reading frame encoding a polypeptide with a 70% amino acid identity with S. cerevisiae proteinase A (SEQ ID. NO: 11). Primers for the identification of PRB1 from P. methanolica were designed based on the alignments between the PRB1 genes of S. cerevisiae (Moehle et al., Mol.Cell. Biol. 7; 4390-4399, 1987), P. pastoris (Gleeson et al., U.S. Patent Number 5,324,660). and Kluyveromyces lactis (Fleer et al., WIPO Publication WO 94/00579). A set of primers, (ZC9126 (SEQ ID NO: 12) and ZC9741 (SEQ ID NO: 13) amplified a fragment of approximately 400 bp of genomic DNA (SEQ ID NO: 14) This product was sequenced and found to encode for a polypeptide with 70% amino acid identity with S. cerevisiae proteinase B (SEQ ID NO: 15) The PRB primer set was then used to identify a genomic clone encompassing the PRB1 gene of P. methanolica Mutations were generated by deletion using restriction enzyme sites available in the genes PEP4 and PRB1 of P. methanolica.The cloned genes were co-related by restriction.The pep4 allele was created by deleting a region of approximately 500 bp between the BamHI and Ncol sites (Figure 3) and including the nucleotides 1 to 393 of the sequence shown in SEQ ID NO: 10. The prbl allele was generated by deleting a region of approximately 1 kbp between the Ncol and EcoRV sites (FIG. 4) and including the sequencing as shown in SEQ ID NO: 14. The cloned PEP4 and PRB1 genes were subcloned into pCZR139, a phagemid vector (pBluescriptR II KS (+), Stratagene, La Jolla, CA) that has an insert of Spel ADE2, of 2.4 kb, to create deletions. In the case of the gene PEP4, the unique BamHI site in pCZRl39 was removed by digestion, by filling and re-ligation. The vector was then linearized by digestion with EcoRI and HindIII, and an EcoRI-HindIII fragment of about 4 kb spanning the PEP4 gene to the linearized vector was ligated to produce the plasmid pCZR142. Then there was a suppression of approximately 500 bp by digesting pZCR142 with Ba HI and Ncol, filling in the ends, and pre-binding the DNA to produce the plasmid pCZR143. The PRB1 gene (Xhol-BamHI fragment of approximately 5 kb) was subcloned into pCZR139, and an intermediate EcoRV-Ncol fragment comprising the sequence shown in SEQ ID NO: 14 was deleted to produce the plasmid pCZR153. Plasmid pCZR143 was linearized with Asp718, which cuts at a single site. The linearized plasmid was introduced into the PMAD11 strain of P. me than ol i ca (a mutant to de2 generated as described in Example 1). The transformants were grown in ADE DS (Table 1) to identify Ade + transformants. Two classes of white Ade + transformants were analyzed. A class appears immediately on the primary transformation plate; the second becomes evident as fast-growing white papids at the edges of colonies of unstable, pink transformants. The Southern blot was used to identify transformants that have undergone desired homologous integration element. 100 ml of the cell paste was discarded from a YEDP plate for 24-48 hours and washed in 1 ml of water. The washed cells were re-dispersed in 400 ml of spheroplast buffer (1.2 M sorbitol, 10 mM Na-citrate, pH 7.5, 10 mM EDTA, 10 mM DTT, 100 mg / ml zymolyase 100 T) and incubated at 37 ° C. C for 10 minutes. Four hundred ml of 1% SDS was used, the cell suspension was mixed at room temperature until it was clear, 300 ml of 5 M potassium acetate was mixed, and the mixture was clarified by microcentrifugation for 5 minutes. 750 ml of clarified lysate were extracted with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1), 600 ml was transferred to a fresh tube, 2 volumes of 100% ethanol were added, and the DNA was precipitated by microcentrifugation for 15 minutes at 4 ° C. The pellet was re-dispersed in 50 ml of TE (10 mM Tris, pH 8.0, 1 mM EDTA) containing 100 mg / ml RNase A. Ten ml of DNA were digested (approximately 100 ng) in a total volume of 100 ml with appropriate enzymes, precipitated with 200 mg of ethanol, and re-dispersed in 10 ml of the DNA loading dye. The DNA was separated in 0.7% agarose gels and transferred to nylon membranes (Nytran N +, Amersham Corp., Arlington Heights, IL) in a semi-dry transfer apparatus (BioRad Laboratories, Richdmond, CA) as recommended by the manufacturer. The transferred DNA was denatured, neutralized and cross-linked to the membrane with UV light using a Stratalinker (Stratagene, La Jolla, CA). To identify the strains with a tandem integration in PEP4, two probes were used. One was an EcoRI-HindIII fragment of 1400 bp from the 3 'end of PEP4. The second was a 2000 bp BamHI-EcoRI fragment from the 5 'end of PEP4. Fragments were detected using chemiluminescence reagents (ECLMR direct labeling equipment, Amersham Corp., Arlington Heighsts, IL). The strains of origin that have a tandem duplication of the wild-type and gene-suppression alleles were grown in YEPD broth overnight to allow the generation of Ade-disconnected strains. These cells were then plated at a density of 2000-5000 colonies per plate in YEPD plates limited in adenine, cultured for 3 days at 30 ° C and 3 days at room temperature. The change at room temperature improved the pigmentation of the rare, pink colonies.The disconnect strains were consistently detected at a frequency of approximately one Ade ~ pink colony per 10 minutes., 000 colonies detected. These strains were detected by attention of wild-type and mutant genes by Southern blotting or by PCR using the primers that spanned the site of suppression. The pep4 strain? ade2-ll was designated PMAD15. The PRB1 gene was then deleted from PMAD15 essentially as described above by transformation with the plasmid pCZR153. Transferences were probed with probes generated by PCR for linear portions of the PRB1 and ADE2 genes. The PRB1 probe was generated by subcloning a Clal-Spel fragment of 2.6 kg of PRBl into the phagemid vector of pBluescriptR II KS (+) to produce pCZR150, and by amplifying the desired region by PCR using the primers ZC447 (SEQ ID NO: 16 and ZC976 (SEQ ID NO: 17) The ADE2 probe was generated by amplifying the ADE2 gene in PCZR139 with the primers ZC9079 (SEQ ID NO: 5) and ZC9080 (SEQ ID NO: 4). The resulting effects of the pep4? and pep4 ?, prbl? mutations in the vacuolar protease activity were determined using the APNE coating assay (Wolf and Fink, J. Ba. ct eri ol. 123: 1150-1156, 1975; Jones, Me th ods On zymol. 194: 428-453, 1991). Protease-competent colonies become leaves in addition to the coating, while mutants deficient in vacuolar protease activity remain white. The colonies PMAD11 and PMAD15 produced a bright red color. In contrast, colonies of PMAD 16 remained white. As long as it is not desired to be a goal, the Pep + phenotype of the pep4 mutant? it may have been a consequence of the phenotypic delay due to the capacity of proteinase B of P. I love it for self-activation. However, the strain pep4 ?, prbl? possesses the deficient protease phenotype, desired.

Example 4 A mutation was generated in ugl? in strain PMAD 11 of P. me than ol i ca. A genomic AUG1 clone and SEQ ID NO: 2 are shown in Figure 2. A deletion allele was made by attaching the 1350 bp AUG1 promoter to the 1600 bp AUG1 terminator and the 3 'untranslated sequence as shown in FIG. Figure 2. A linear DNA construct comprising the deletion allele of a selected ADE2 marker was electroporated into PMAD11, and Ade + transformants were selected essentially as described in Example 3. The homologous recombinants were identified by Southern blotting. . Ade + auxotrophs in which the AUG1 locus was doubled were cultured in YEPD broth overnight, then transferred to YEPD plates. The pink colonies were harvested and selected for disconnection of the wild-type locus by Southern blots and PCR. The augl mutant strain, designated PMAD12, grows poorly in minimal methanol broth. In minimal methanol plates, PMAD12 exhibited a slight growth effect in relation to wild-type cells. These data suggest that the Augl protein plays an important role in the assimilation of methanol, particularly in the liquid medium, but that P. me olica possesses a second alcohol-oxidase activity.

Example 5 A second alcohol oxidase gene was identified in P. I was using the PCR-specific alcohol-oxidase primers to amplify the genomic DNA of an augl mutant strain. A weak 600 bp signal was detected, which was re-amplified and subjected to DNA sequencing (SEQ ID NO: 3). The translation of this sequence and the alignment with the Augl sequence showed an identity of 83% with a corresponding relationship with the Augl protein. The PCR fragment was then used as a probe to identify a full-length clone for this second alcohol-oxidase gene, which was designated AUG2 (Figure 5). A disordered allele of the AUG2 gene was constructed as shown in Figure 5 by deleting the two BglII fragments within the open reading frame. The aug2 mutant allele? was combined with the ADE2 selectable marker, and the resulting linear construct was introduced into strains PMAD11, PMAD12 and (a ugl?) of P. me than ol i ca. The resulting deletion mutants, PMAD13 (a ug2A) and PMAD14 (a ugl? A ug2?) Were selected and identified essentially as described in Examples 3 and 4. These isogenic strains were cultured in minimal methanol broth and methanol plates. minimum. The double mutant was unable to grow in minimal methanol medium of any kind, indicating that the AUG1 and AUG2 genes are the only alcohol oxidase genes of P. me thanol i ca. While the strain to ugl? grew poorly in the minimum methanol broth, the mutant at ug2? in a way comparable to the wild type cell. These data suggest that the Augl protein plays a major role in the use of methanol during growth in liquid cultures. When planted, the differential growth between the mutants to ugl? and to ug2? It was much less pronounced. Examination of the total cell extracts by SPD-PAGE showed that the AUG2 protein was strongly induced in cells to ugl? planted, but poorly grown in cultures in shake flasks or in cells grown under fermentation conditions induced by methanol.

Example 6 A human glutamic decarboxylase (GADes) expression vector was constructed by inserting the cDNA coding for GAD55 (Karlsen et al., Proc. Nati, Acad. Sci. USA 88: 8337-8341, 1991 ) as an EcoRI-Xbal fragment in the EcoRI-Spel sites of plasmid pCZR134 (Figure 6). The resulting expression vector, pCZR137, comprised the AUG1 promoter and the ADE2 selectable marker and terminator. Plasmid pCZR137 was digested with Notl and used to transform PMAD16 to Ade +. A thousand stable Ade + transformants were detected for the expression of GAD65 in minimal methanol plates using a nitrocellulose coating, colony lysis and western blots technique essentially as described by Wuestehube et al., Gen e ti cs 142: 393-406, 1996. The transformants were patched in gratings of 50 minimal plates lacking adenine, cultured for 24 hours. hours at 30 ° C, were plated on replicate plates in minimal methanol plates, coated with nitrocellulose and incubated for at least 48 hours at 30 ° C. The filters were removed from the plates, dried and the side of the colony was placed upward for 30 minutes at room temperature on filter paper saturated with lysis buffer (0.1% SDS, 0.2N NaOH, 35 mM DTT). The filter debris was rinsed under a stream of distilled water, and the filters were neutralized by a 5 minute incubation in 0.1 M acetic acid. The filters were then blocked in TTBS-NFM (20 mM Tris, pH 7.4, I was 160 mM, 0.1% Tween 20, 5% non-fat milk) and incubated in TTBS-NDM containing the monoclonal antibody GAD65_pecific of human GAD65 (Chang and Gottlieb, J. Neuros ci. 8_: 2123-2130, 1988 ). A goat anti-mouse antibody conjugated with horseradish peroxidase was used to detect GAD65-specific immune complexes, which were visualized with commercially available chemiluminescence reagents (ECLRM; Amersham Inc., Arlington Heights, IL) according to conventional techniques. Ninety percent of the transformants were found to express GAD65. Forty-six strains that appeared to express the highest levels of G Ü65 were re-titrated by SDS-PAGE / western analysis. Forty-four of these strains appeared to have identical levels of GAD65. Analysis by Southern blots (essentially as described in Example 3) indicated that these strains had an individual copy of the GAD 55 'expression cassette. Two strains appeared to have high levels of GAD65. Both of these strains exhibited slow growth in minimal methanol broth, and the analysis of the genomic DNA of these strains by PCR using primers specific for AUG1 revealed that these strains were a ugl? , indicating that trans-placement of the wild-type AUG1 gene by the G Dßs expression cassette had occurred. The strain to ugl? which makes the apparently higher levels of GADes, PGAD4-2, was cultured under high cell density fermentation conditions in a BioFlow 3000 fermentor (New Brunswick Scientific Co., Ind., Edison, NJ). An inoculum was generated by dispersing cells from a YEPD plate for 2 days in 250 ml of YEPD broth, and the culture was shaken vigorously overnight in a 1 liter baffled flask at 30 ° C. The fermentation vessel was charged with 2.5 liters of medium containing 57.8 g of (NH4) 2S04 '46.6 g of KCl, 30.8 g of MgSO4 7H20, 8.6 g of CaS0 2 H20, 2.0 g of NaCl, and 10 ml of antifoam. After purification by autoclaving and cooling the vessel to a working temperature of 29 ° C, 350 ml of 50% glocosa, 210 ml of 30% sodium hexametaphosphate (phosphate crystal), and 250 ml of trace elements were added. (containing, per liter, 27.8 g of FeS04 7H20, 0.5 g of CuS04 5H20, 1.09 g of Zncl2, 1.35 g of MnS0 H20, 0.48 g of CoCl2, 6H20, 0.24 g of Na2Mo0 2H20, 0.5 g of H3B03, 0.08 g of KI, 5 mg of biotin, 0.5 g of thiamin, and 2.5 ml of H2SO4). The pH of the fermenter was adjusted to 5.0 and controlled automatically with 0% NH 0H and 10% H3P0. Aeration was initially provided with compressed air provided at a flow rate of 5 liters / minute and an agitator speed of 300 rpm. After the dissolved oxygen was adjusted to 100%, the cell inoculum was added. The dissolved oxygen control was adjusted to maintain 30% saturation within and in the agitation range of 300-800 rpm. Oxygen demand above 800 rpm activated automatic supplementation with pure oxygen. The phase of the growth lot was characterized by a stable increase in demand over a period of 24-36 hours. After glucose depletion, the demand for oxygen fell rapidly, and a glucose feed (containing 750 g of glucose, 110 g of (NH4) 2S04, and 278 ml of trace elements) was started at a speed of 0.4% glucose / hour. After 25 hours, the transition to methanol induction of the AUG1 promoter was made with a mixed feed of glucose (0.2% / hour) and methanol (0.2% / hour) for 5 hours. A final mixed feed of methanol (0.1% glucose / hour, 0.4% methanol / hour) was run for 25 hours. Strong expression of GAÜ65 was induced by the addition of methanol. The level of expression of GADßs was calculated to be about 500 mg / L in a final cell mass of 170 grams of cell paste / 1.

Example 5 (7) A strain of P. methanol i ca deficient in vacuolar protease (pep4? Prbl?) That was genetically suppressed for the main alcohol reductase (a ugl?) Was prepared from the strain PMAD16 (ade-2). -d-Il pep4?) prbl? ). This strain was transformed to Ade + with an interruption plasmid AUG1 pCZR140-6 which had been linearized with the restriction enzyme Asp718I. Plasmid pCZR140-6 is a vector based on Bluescript® (Stratagene Clonig Systems, La Jolla, CA) - containing the ADE2 gene from P. me than ol i ca and an AUG1 mutant in which the complete open reading frame between the promoter and terminator region has been suppressed (Figure 7). The unstable Ade + transformants (which arise when the transformation DNA is formed in a circle and the subsequent episomal propagation of the plasmid due to the presence of an-ARS in the ADE2 marker) were identified by slow growth and pink color in the ADE DS medium. The cells that have integrated the circular episome by homologous recombination produced a white porridge, of fast growth in the edges of the pink colonies, of slow growth. The Ade + stable slurry of the PMAD16 cells transformed with the plasmid pCZR140-6 were isolated, and the genomic DNA was prepared. The DNA was digested with EcoRI and subjected to Southern blot analysis. A probe corresponding to the AUG1 promoter region was generated by PCR using the oligonucleotide primers ZC9081 (SEQ ID NO: 18) and ZC9084 (SEQ ID NO: 19) and, as a primer, a plasmid containing the AUG1 promoter fragment from pZCR134. Probing of the transfer revealed that four of ten Ade + stable porridges examined had undergone homologous recombination of the AUG1 disruption plasmid in the AUG1 promoter region. These four colonies were placed in multiple plates of a non-selective medium (YEPD) to allow the growth of both Ade + and Ade colonies. "(In the YEPD, the Ade colonies" developed a pink color due to starvation by adenine and expression. Subsequent phenotype to de2 (pink) The integrated AUG1 interruption plasmid spontaneously undergoes mitotic homologous recommendation, effectively disconnecting the plasmid from the genome.These "disconnected" cells can be detected because they develop in pink colonies in nonselective media. disconnection of the interruption plasmid au gl either restores the wild-type AUG1 allele or leaves the allele of interruption to ugl? in the AUG1 locus, depending on the recombination site). Ade disconnection colonies were detected by PCR using primers ZG10,635 (SEQ ID NO: 20) and ZG14, 199 (SEQ ID NO: 21) for interrupted or disordered strains to ugl.10 of 15 strains detected produced a product of PCR of 600 base pairs, indicating that they have retained the allele to ugl.The remaining 5 strains detected yielded a 2.1 kb wild-type AUG1 PCR product.The subsequent growth test in minimal methanol broth revealed that the 10 strains A putative ugl grew slowly in this medium while the 5 putative AUG1 cells grew well in this medium.This phenotype is characteristic of the ugl mutants.One of these colonies, isolate # 3 was given the designation of strain PMAD 18 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.

SEQUENCE LIST < 110 > ZymoGenetics, Inc. < 120 > CRO OSOMICA MUTAGENESIS IN PICHIA METANOLICA < 130 > 97-70PC < 150 > 60 / 058,822 < 151 > 1997-09-15 < 150 > 09 / 001,141 < 151 > 1997-12-30 < 160 > twenty-one < 170 > FastSEQ for Windows Version 3.0 < 210 > 1 < 211 > 3077 < 212 > DNA < 213 > Pichia methanolica <; 400 > 1 cagctgctct gctccttgat tcgtaattaa tgttatcctt ttactttgaa ctcttgtcgg tccccaacag ggattccaat cggtgctcag cgggatttcc catgaggttt ttgacaactt 1 tattgatgct gcaaaaactt ttttagccgg gtttaagtaa ctgggcaata tttccaaagg 1 ctgtgggcgt tccacactcc ttgcttttca taatctctgt gtattgtttt attcgcattt 2 tgattctctt attaccagtt atgtagaaag atcggcaaac aaaatatcaa cttttatctt 3 gaacgctgac ccacggtttc aaataactat cagaactcta tagctatagg ggaagtttac 3 tgcttgctta aagcggctaa aaagtgtttg gcaaattaaa caagtaggaa aaagctgtga 4 ctcctgtaaa gggccgattc gagcctaaaa gacttcgaaa acagtgacta ttggtgacgg 4 aaaattgcta aaggagtact agggctgtag taataaataa tggaacagtg gtacaacaat 5 aaaagaatga cgctgtatgt cgtagcctgc acgagtagct cagtggtaga gcagcagatt 6 gcaaatctgt tggtcaccgg ttcgatccgg tctcgggctt ccttttttgc tttttcgata 6 tttgcgggta ggaagcaagg tctagttttc gtcgtttcgg atggtttacg aaagtatcag 7 ccatgagtgt ttccctctgg ctacctaata tatttattga tcggtctctc atgtgaatgt 7 gttcggcttt ttctttccaa cagctcgtaa atgtgcaaga aatatttgac tccagcgacc 8 tttcagagtc aaattaattt tcgctaacaa tttgtgtttt tctg gagaaa cctaaagatt 9 gtcgaatcaa taactgataa catctttaaa tcctttagtt aagatctctg cagcggccag 9 tagcatattc tattaaccaa acaggcatca catcggaaca ttcagaatgg actcgcaaac 10 tgtcgggatt ttaggtggtg gccaacttgg tcgtatgatc gttgaagctg cacacagatt 10 gaatatcaaa actgtgattc tcgaaaatgg agaccaggct ccagcaaagc aaatcaacgc 11 tttagatgac catattgacg gctcattcaa tgatccaaaa gcaattgccg aattggctgc 12 caagtgtgat gttttaaccg ttgagattga acatgttgac actgatgcgt tggttgaagt 12 actggcatca tcaaaaggca aaatcttccc atcaccagaa actatttcat tgatcaaaga 13 taaatacttg caaaaagagc atttgattaa gaatggcatt gctgttgccg aatcttgtag 13 tgttgaaagt agcgcagcat ctttagaaga agttggtgcc aaatacggct tcccatacat 14 gctaaaatct agaacaatgg cctatgacgg aagaggtaat tttgttgtca aagacaagtc 15 atatatacct gaagctttga aagttttaga tgacaggccg ttatacgccg agaaatgggc 15 tccattttca aaggagttag ctgttatggt tgtgagatca atcgatggcc aagtttattc 16 ctacccaact gttgaaacca tccaccaaaa caacatctgt cacactgtct ttgctccagc 16 tagagttaac gatactgtcc aaaagaaggc ccaaattttg gctgacaacg ctgtcaaatc 17 tttcccaggt gctg gtatct ttggtgttga aatgttttta ttacaaaatg gtgacttatt 18 attgccccaa agtcaacgaa gacctcacaa ttctggtcac tataccatcg acgcttgtgt 18 cacctcgcaa tttgaagctc atgttagggc cattactggt ctacccatgc cgaagaactt 19 cacttgtttg tcgactccat ctacccaagc tattatgttg aacgttttag gtggcgatga 19 gcaaaacggt gagttcaaga tgtgtaaaag agcactagaa actcctcatg cttctgttta 20 cttatacggt aagactacaa gaccaggcag aaaaatgggt tagtttctca cacattaata 21 atcaatgact gactgtgagc gtagattaca ggtacgacta ttacatagaa acagcatccc 21 cagtacacta tctcgaagaa cagattccat tccgggcact tcaagcaagc cattagtcgg 22 tgtcatcatg ggttccgatt cggacctacc agtcatgtct ctaggttgta atatattgaa 22 gcaatttaac gttccatttg aagtcactat cgtttccgct catagaaccc cacaaagaat 23 ggccaagtat gccattgatg ctccaaagag agggttgaag tgcatcattg ctggtgctgg 24 tggtgccgct catttaccgg gaatggttgc ggcgatgacg ccgctgcctg ttattggtgt 24 ccctgttaaa ggctctactt tggatggtgt tgattcacta cactccatcg ttcaaatgcc 25 aagaggtatt cctgttgcta ctgtggctat taacaatgct actaacgctg ccttgctagc 25 tatcacaatc ttaggtgccg gcgatccaaa tacttgtctg caatg gaagt ttatatgaac 26 aatatggaaa atgaagtttt gggcaaggct gaaaaattgg aaaatggtgg atatgaagaa 27 catacaagaa tacttgagta gtagaacctt ttatatttga tatagtactt actcaaagtc 27 ttaattgttc taactgttaa tttctgcttt gcatttctga aaagtttaag acaagaaatc 28 ttgaaatitc tagttgctcg taagaggaaa cttgcattca aataacatta acaataaatg 28 acaataatat attatttcaa cactgctata tggtagtttt ataggtttgg ttaggatttg 29 agatattgct agcgcttatc attatcctta attgttcatc gacgcaaatc gacgcatttc 30 cacaaaaatt ttccgaacct gtttttcac tctccagatc ttggtttagt atagcttttg 30 acacctaata cctgcag 30 < 210 > 2 < 211 > 3385 < 212 > DNA < 213 > Methane leg < 400 > 2 gaattcctgc agcccggggg atcgggtagt ggaatgcacg gttataecca aagtgtagta ctccaaataa gccggactga aaggttttag gagtctgttt gtttgttcat gtgeatcatt 1 ccctaatctg ttaacagtct cggagtatac aaaaaagtaa gtcaaatatc aaggtggccg 1 ggggcagcat egagactega gatggtacat acttaaaagc tgccatattg aggaacttca 2 tgtttttaga aagttttatc attaaaagac gattgttgte acaaaa gtt gtgectacat 3 aaactcaaat taatggaaat agcctgtttt gaaaaataca cttettaag tactgacaaa 3 gttttgttaa atgactatcg aacaagccat gaaatagcac atttctgcca gtcactttta 4 acactttcct gcttgctggt tgactctcct catacaaaca cccaaaaggg aaactttcag 4 tgtggggaca cttgacatct cacatgcacc ccagattaat ttccccagac gatgcggaga 5 caagacaaaa caaccctttg tcctgctctt ttctttctca caccgcgtgg gtgtgtgcgc 6 aggcaggcag gcaggcagcg ggctgcctgc catctctaat cgctgctcct cccccctggc 6 ttcaaataac agcctgctgc tatctgtgac cagattggga cacccccctc ccctccgaat 7 gatccatcac cttttgtcgt actccgacaa tgatccttcc ctgtcatctt ctggcaatca 7 gctccttcaa taattaaatc aaataagcat aaatagtaaa atcgcataca aacgtcatga 8 aaagttttat ctctatggcc aacggatagt ctatctgctt aatt ccatcc actttgggaa 9 ccgctctctc tttaccccag attctcaaag ctaatatctg ccccttgtct attgtccttt 9 ctccgtgtac aagcggagct tttgcctccc atcctcttgc tttgtttcgg ttattttttt 10 ttcttttgaa actcttggtc aaatcaaatc aaacaaaacc aaaccttcta ttccatcaga 10 tcaaccttgt tcaacattct ataaatcgat ataaatataa ccttatccct cccttgtttt 11 ttaccaatta atcaatcttc aaatttcaaa tattttctac ttgctttatt actcagtatt 12 aacatttgtt taaaccaact ataactttta actggcttta gaagttttat ttaacatcag 12 catctttatt tttcaattta tattaacgaa atctttacga attaactcaa tcaaaacttt 13 tacgaaaaaa aaatcttact attaatttct caaaatggct attccagatg aatttgatat 13 tattgttgtc ggtggtggtt ccaccggttg tgctcttgct ggtagattag gtaacttgga 14 cgaaaacgtc acagttgctt taatcgaagg tggtgaaaac aacatcaaca acccatgggt 15 ttacttacca ggtgtttatc caagaaacat gagattagac tcaaagactg ctacttttta 15 ctcttcaaga ccatcaccac acttgaacgg tagaagagct attgttccat gtgctaacat 16 ggttcttcca cttgggtggt tcaacttctt gatgtacacc agagcctctg cctccgatta 16 cgatgattgg gaatctgaag gttggactac cgatgaatta ttaccactaa tgaagaagat 17 tgaaacttat caaa gaccat gtaacaacag agaattgcac ggtttcgatg gtccaattaa 18 ggtaactata ggtttcattt cttatccaaa cggtcaagat ttcattagag ctgccgaatc 18 tcaaggtatt ccatttgttg atgatgctga agatttgaaa tgttcccacg gtgctgagca 19 ctggttgaag tggatcaaca gagacttagg tagaagatcc gattctgctc atgcttacat 19 tcacccaacc atgagaaaca agcaaaactt gttcttgatt acttccacca agtgtgaaaa 20 gattatcatt gaaaacggtg ttgctactgg tgttaagact gttccaatga agccaactgg 21 ttctccaaag acccaagttg ctagaacttt aagcaaatta caaggctaga ttgtttcttg 21 tggtactatc tcatcaccat tagttttgca aagatctggt atcggttccg ctcacaagtt 22 gagacaagtt ggtattaaac cttaccaggt caattgttga acttccaaga gttggtatga 22 tcactactgt ttcttcactc cataccatgt caagccagat actccatcat tcgatgactt 23 tgttagaggt gataaagctg ttcaaaaatc tgctttcgac caatggtatg ctaacaagga 24 tggtccatta accactaatg gtattgaggc aggtgttaag attagaccaa ctgaagaaga 24 attagccact gctgatgacg aattcagagc tgcttatgat gactactttg gtaacaagcc 25 agataagcca ttaatgcact actctctaat ttctggtttc ttggtgacc acaccaagat 25 tccaaacggt aagtacatgt gcatgttcca cttcttggaa tatcca TTCT ccagaggttt 26 cgttcacgtt gtttctccaa acccatacga tgctcctgac tttgatccag gtttcatgaa 27 cgatccaaga gatatgtggc caatggtttg gtcttacaag aagtccagag aaactgccag 27 aagaatggac tgttttgccg gtgaagttac ttctcaccac ccacactacc catacgactc 28 accagccaga gctgctgaca tggacttgga aactactaaa gtccagacca gcttatgctg 28 ctttactgct aacttgtacc acggttcatg gactgttcca attgaaaagc caactccaaa 29 gaacgctgct cacgttactt ctaaccaagt tgaaaaacat cgtgacatcg aatacaccaa 30 ggaggatgat gctgctatcg aagattacat cagagaacac actgaaacca catggcattg 30 tcttggtact tgttcaatgg ctccaagaga aggttctaag gttgtcccaa ctggtggtgt 31 tgttgactcc agattaaacg tttacggtgt tgaaaagttg aaggttgctg atttatcaat 31 ttgcccagat aatgttggtt gtaacactta ctctactgct ttgttaatcg gtgaaaaggc 32 ttctacctta gttgctgaag acttgggcta ctctggtgat gctttgaaga tgactgttcc 33 aaacttcaaa ttgggtactt atgaagaagc tggtctagct agattctagg gctgcctgtt 33 tggatatttt tataattttt gagagt 33 < 210 > 3 < 211 > 586 < 212 > DNA < 213 > Methanolamine < 400 > 3 gatctgátgc tgcgcatgct tacattcacc caactatgag aaacaagtca tgatcaette aacttatact cactaaggct gataaagtta tggagttgca taattgaaga gctggtattc 1 ttccaaacca aagttgttcc ttgaacccag aaaagccggc tgecaagate tacaaggcta 1 gaaagaaat cattctatcc tgtggtacaa tttctacccc gttggtccta caaagatctg 2 gtattggctc agetcataaa ttaagacagg caggcataaa accgatcgtt gacttgccag 3 gagttggtat gaattecaa gatcactact gctttttcac cccataccat gtcaagccag 3 atactccttc ttttgatgac tttgccagag gtgataagac tgttcaaaaa tcagcttttg 4 atcaatggta tgctaacaaa gatggtcctt taaccactaa cggtattgaa gctggtgtta 4 agattagacc aactgctgaa gaactggcta ctgctgatga agatttecaa ctaggctacg 5 cttcttactt tgaaaacaag ccagataaac cattgatgca ttaetc 5 < 210 > 4 < 211 > 38 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide primer < 400 > 4 tgatcaecta ggactagtga caagtaggaa ctcctgta < 210 > 5 < 211 > 39 < 212 > DNA < 213 Artificial Sequence < 220 > < 223 > Oligonucleotide primer < 400 > 5 cagctgccta ggactagttt cctcttacga gcaactaga < 210 > 6 < 211 > 17 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide Primer < 400 > 6 tggttgaagt ggatcaa 1 < 210 > 7 < 211 > 17 < 212 > DNA 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide Primer < 400 > 7 gtgtggtcac cgaagaa 1 < 210 > 8 < 211 > 17 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide Primer < 400 > 8 acctcccagt aagcctt 1 < 210 > 9 < 211 > 17 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide primer < 221 > variation < 222 > (1 ) . . . (17) < 223 > n is any nucleotide < 400 > 9 ttyggnaart tygaygg < 210 > 10 < 211 > 421 < 212 > DNA < 213 > Pichia methane! Ica < 220 > < 221 > CDS < 222 > (2). (421) < 400 > 10 g gaa ggt aac gtt tct cag gat act tta gct tta ggt gat tta gtt att 4 Glu Gly Asn Val Ser Gln Asp Thr Leu Ala Leu Gly Asp Leu Val He 1 5 10 15 cea aaa caa gac ttt gee gaa gct act tct gag cea ggt tta gca ttc 9 Pro Lys Gln Asp Phe Wing Glu Wing Thr Ser Glu Pro Gly Leu Wing Phe 20 25 30 gca ttt ggt aaa ttt gat ggt att tta ggt tta gct tac gat age att 14 Wing Phe Gly Lys Phe Asp Gly He Leu Gly Leu Ala Tyr Asp Ser He 35 40 45 teg gtc aac aag att gtt ect ect att tat aat gct tta aac ttg ggt 19 Ser Val Asn Lys He Val Pro Pro He Tyr Asn Ala Leu Asn Leu Gly 50 55 60 tta tta gat gaa ect caa ttt gee ttc tac cta ggt gat act aac acc 2 Leu Leu Asp Glu Pro Gln Phe Wing Phe Tyr Leu Gly Asp Thr Asn Thr 65 70 75 80 aat gaa gaa gat ggt ggt ctt gee act ttt ggt ggt gtt gat gag tec 2 Asn Glu Glu Asp Gly Gly Leu Wing Thr Phe Gly Gly Val Asp Glu Ser 85 90 95 aag tat act ggt aaa gtt htgg tta cea gtc aga aga aag gct tac 3 Lys Tyr Thr Gly Lys Val Thr Trp Leu Pro Val Arg Arg Lys Ala Tyr 100 105 110 tgg gaa gtt tea tta gac ggt att tea tta ggt gat gaa tac gcg cea 3 Trp Glu Val Ser Leu Asp Gly He Ser Leu Gly Asp Glu Tyr Ala Pro 115 120 125 tta gaa ggc cat gga gct gcc gt att gat ggt acc 42 Leu Glu Gly His Gly Ala Ala He Asp Thr Gly Thr 130 135 140 < 210 > 11 < 211 > 140 • < 212 > PRT < 213 > Pichia methane! i ca < 400 > 11 Glu Gly Asn Val Ser Gln Asp Thr Leu Wing Leu Gly Asp Leu Val He 1 5 10 15 Pro Lys Gln Asp Phe Wing Glu Wing Thr Ser Glu Pro Gly Leu Wing Phe 20 25 30 Wing Phe Gly Lys Phe Asp Gly He Leu Gly Leu Ala Tyr Asp Ser He 35 40 45 Ser Val Asn Lys He Val Pro Pro He Tyr Asn Ala Leu Asn Leu Gly 50 55 60 Leu Leu Asp Glu Pro Gln Phe Wing Phe Tyr Leu Gly Asp Thr Asn Thr 65 70 75 80 Asn Glu Glu Asp Gly Gly Leu Wing Thr Phe Gly Gly Val Asp Glu Ser 85 90 95 Lys Tyr Thr Gly Lys Val Thr Trp Leu Pro Val Arg Arg Lys Wing Tyr 100 105 110 Trp Glu Val Ser Leu Asp Gly He Ser Leu Gly Asp Glu Tyr Ala Pro 115 120 125 Leu Glu Gly Hi s Gly Ala Ala He Asp Thr Gly Thr 130 135 140 < 210 > 12 < 211 > 17 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide primer < 400 > 12 atgtcaacac atttacc < 210 > 13 < 211 > 17 < 212 > ? DN < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide primer < 221 > "variation < 222 > (D .. C17) < 223 > n is any nucleotide < 400 > 13 cayggnacnc aytgygc 1 < 210 > 14 < 211 > 368 < 212 > DNA < 213 > Pichia Methanolica < 220 > < 221 > CDS < 222 > ü) ... (366) < 221 > variation < 222 > (1 ) . . . (368) < 223 > n is any nucleotide < 400 > 14 ggg tcc gna ene atg gtg ttt cta aga att gee falls att gtt gee gtc 48 Gly Ser Xaa Met Val Phe Leu Arg He Ala His Val Val Val 1 5 10 15 aaa gtt tta aga tct aac ggt tea ggt tct atg ecc gat gtt gtc aag 96 Lys Val Leu Arg Ser Asn Gly Ser Gly Ser Met Pro Asp Val Val Lys 20 25 30 ggt gtt gaa tat gct ecc aat gct falls ctt gcg gaa gee aag gct aac 144 Gly Val Glu Tyr Ala Pro Asn Ala His Leu Ala Glu Ala Lys Ala Asn. 35 40 45 aag agt ggt ttt aaa ggt tct ac gcg aac atg tea tta ggt ggt ggt 192 Lys Ser Gly Phe Lys Gly Ser Thr Ala Asn Met Ser Leu Gly Gly Gly 50 - 55 60 aaa tct cea gct tta gat atg tct gtt aac gct ect gtt aaa gca ggt .240 Lys Ser Pro Wing Leu Asp Met Ser Val Asn Wing Pro Val Lys Wing Gly 65 70 75 80 tta drops ttt gcc gtt acc gct ggt aac gat aac act gat gca tgt aac 28 Leu His Phe Wing Val Thr Wing Gly Asn Asp Asn Thr Asp Wing Cys Asn 85 90 95 tat tct cea gcc act act gaa aat act gtc act gtt gtt gct tct act 33 Tyr Ser Pro Wing Thr Thr Glu Asn Thr Val Thr Val Val Ala Ser Thr 100 - 105 110 tta tct gat tcg aga gct gac atg tct aac te 36 Leu Ser Asp Ser Arg Ala Asp Met Ser Asn 115 120 < 210 > 15 < 211 > 122 < 212 > PRT < 213 > Pichia Metanolica < 220 > < 221 > VARIANT < 222 > (D ... Ü22) < 223 > Xaa is any amino acid < 400 > 15 Gly Ser Xaa Met Val Phe Leu Arg He Ala His Val Val Val 1 5 10 15 Lys Val Leu Arg Ser Asn Gly Ser Gly Ser Met Pro As Val Val Lys 20 25 30 Gly Val Glu Tyr Ala Pro Asn Ala His Leu Ala Glu Ala Lys Ala Asn 35 -40 45 Lys Ser Gly Phe Lys Gly Ser Thr Ala Asn Met Ser Leu Gly Gly Gly 50 55 60 Lys Ser Pro Ala Leu Asp Met Ser Val A = n Ala Pro Val Lys Ala Gly 65 70 75 80 Leu His Phe Wing Val Thr Wing Gly Asn Asp Asn Thr Asp Wing Cys Asn 85 90 95 Tyr Ser Pro Wing Thr Thr Glu Asn Thr Val Thr Val Val Wing Ser Thr 100 105 110 Leu Ser Asp Ser Arg Wing Asp Met Ser Asn 115 120 < 210 > 16 < 211 > 17 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide Primer < 400 > 16 taacaatttc acacagg < 210 > 17 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide Primer < 400 > 17 cgttgtaaaa cgacggcc < 210 > 18 < 211 > 48 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide Primer < 400 > 18 tcgatggatc cggaattcgt taaataaaac ttctaaagcc agttaaaa 4 < 210 > 19 < 211 > 33 < 212 > DNA 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide primer < 400 > 19 ctagcaagat ctccggggga tcgggtagtg gaa 3 < 210 > 20 < 211 > 22 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 Oligonucleotide Primer 400 > 20 ccaactataa cttttaactg ge 2 < 210 > 21 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide primer < 400 > 21 aaaagatatc caactacatg 2 It is noted that in relation to this date, the best method known by the applicant to carry out the present invention is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property:

Claims (13)

1. CLAIMS 1. A method for altering a chromosomal locus of Pi chi a me than ol i ca cells, characterized in that it comprises: (a) selecting an objective chromosomal locus of the cells; (b) providing a population of P. me tha n ol i ca cells each comprising a chromosomal copy of the locus, wherein the cells are auxotrophic for adenine; (c) introducing into the provided cells a linear DNA construct comprising (i) a segment comprising a portion of the target chromosomal locus in which at least one pair of nucleotides is altered, and (ii) a selectable marker that complements the adenine auxotrophy: (d) culturing the cells of step (c) under conditions that are selective for the presence in cells of the selectable marker; (e) identifying the subset of the cultured cells in which the segment of the DNA construct and the selectable marker have been chromosomally integrated by homologous recombination, the recombination which results in tandem duplication of the target chromosomal locus; (f) culturing the identified subset of cells under conditions wherein the prototrophic cells for adenine grow and exhibit a first phenotype, and the auxotrophic cells for adenine grow and exhibit a second phenotype; (g) recovering cells that are auxotrophic for adenine; and (h) identifying a subset of auxotrophic cells in which the segment of the DNA construct is chromosomally integrated, and whereby the target chromosomal locus is altered.
2. 2. The method according to claim 1, characterized in that a plurality of nucleotide pairs of the chromosomal locus portion is altered in the segment.
3. 3. The method according to claim 1, characterized in that from 1 kbp to 2 kbp of the chromosomal locus portion is altered in the segment.
4. 4. The method according to claim 1, characterized in that at least one pair of nucleotides is altered by deletion.
5. 5. The method according to claim 1, characterized in that the target chromosomal locus encodes a pro-tease.
6. 6. The method according to claim 5, characterized in that the protease is proteinase A or proteinase B.
7. 7. The method according to claim 1, characterized in that the objective chromosomal locus qualifies for an alcohol oxidase.
8. 8. The method according to claim 1, characterized in that steps (a) and up (h) are repainted, whereby two chromosomal loci are altered.
9. 9. The method according to claim 8, characterized in that one of the two chromosomal loci codes for a protease. and a second of the chromosomal loci codes for an alcohol oxidase.
10. 10. The method according to claim 1, characterized in that the objective chromosomal locus is a nutritional chain.
11. 11. The method according to claim 1, characterized in that the selectable marker comprises nucleotides 407-2851 of SEQ ID NO: 1.
12. 12. The method according to claim 1, characterized in that the target chromosomal locus is a gene selected from of the group consisting of the genes PEP4 r PRB1, and A UG2.
13. 13. A Pi chi cell is characterized in that it is produced by the method of any of claims 1-12. CHROMOSOMAL MUTAGENESIS IN P I CHIA METHANOLICA SUMMARY OF THE INVENTION Methods are described for altering a selected chromosomal locus in P. me thanol i ca cells and cells comprising these altered loci. A linear DNA construct comprising (i) a segment comprising a target locus portion in which at least one nucleotide pair is altered and (ii) selectable marker that complements the adenine auxotrophy that is introduced into auxotrophic cells for adenine. The cells are cultured under selective conditions, in which the linear DNA construct has been integrated chromosomally by homologous recommendation are identified. Then, the cells are cultured under conditions whereby the auxotrophic cells for adenine can be identified, and are identified in a subset of these cells in which the altered locus has been chromosomally integrated.