IE83234B1 - DNA fragment encoding an AOX - Google Patents

Description

PATENTS ACT 1992 DNA FRAGMENT ENCODING AN AOX RESEARCH CORPORATION TECHNOLOGIES, INC.

This invention relates to the field of recombinant DNA technology. In one aspect, this invention relates to the integrative transformation of yeast. In another aspect, the present invention relates to site-directed mutation of yeast.

In yet another aspect, the present invention relates to novel DNA sequences. In a further aspect, the present invention relates to novel organisms.

Background As recombinant DNA technology has developed in recent years, the controlled production by microorganisms of an enormous variety of useful polypeptides has become possible. Many eukaryotic polypeptides, such as for example, human growth hormone, human insulin leukocyte interferons, and human proinsulin have already been produced by various microorganisms. The continued application of techniques already in hand is expected in the future to permit production by ndcroorganisms of" a variety of other useful polypeptide products.

A basic element frequently employed in recombinant technology is the extrachromosomal, where plasmid, which is double-stranded DNA found in some microorganisms. plasmids have been found to naturally occur in microorganisms, they are often found to occur lJ1 multiple copies per cell. In addition to naturally occurring plasmids, a variety of man—made plasmids, or hybrid Vectors, have been prepared. Unfortunately, it is not always possible for the host cell to maintain the plasmid. Instead, the plasmid is lost as the organism reproduces and passes through Methods for the stable introduction of foreign DNA into suitable host organisms are several generations of growth. therefore of great interest and potentially of great value.

Up to now, commercial efforts employing recombinant DNA technology for producing various polypeptides have centered on Escherichia coli as a host organism. However, in some situations E. coli may prove to be unsuitable as a host.

For example, E. coli contains a number of toxic pyrogenic factors that must be eliminated from any polypeptide useful as a pharmaceutical product. The efficiency with which this purification can be achieved will, of course, vary with the particular polypeptide. In addition, the proteolytic activities of .E. coli can seriously limit yields of some useful products. Furthermore, a number of heterologous gene products which have been produced in E. coli have been found to be insoluble considerations have led to increased interest in alternate hosts. the production of polypeptide products is appealing. produced in form. These and other In particular, the use of eukaryotic organisms for The availability of means for the production. of e.g., could provide significant advantages relative to the use of polypeptide products in eukaryotic systems, yeast, prokaryotic systems such as E. coli for the production of polypeptides encoded by recombinant DNA. Yeast has been employed in large scale fermentations for centuries, as compared to the relatively recent advent of large scale E. coli fermentations. Yeast can generally be grown to higher cell densities than bacteria and are readily adaptable to continuous fermentation processing. In fact, growth of yeast such as Pichia pastoris to ultra—high cell densities, i.e., cell densities in excess of 100 g/L, in U.S. 4,414,329 (assigned to Phillips Petroleum Co.).

Additional advantages of yeast hosts include the fact that many critical functions of the organism, e.g., is disclosed by wegner oxidative phosphorylation, are located within organelles, and hence are not exposed to possible deleterious effects caused by the organisms production of polypeptides foreign to the wild-type host cells. As a eukaryotic organism, yeast may prove capable of glycosylating expressed polypeptide products, which may prove of value where such glycosylation is important to the bioactivity of the polypeptide product. It is also possible that as a eukaryotic organism, yeast will exhibit the same codon preferences as higher organisms, thus tending toward more efficient production of expression products from mammalian genes or from complementary DNA (CDNA) obtained by reverse transcription from, mammalian mRNA. for example, The development of poorly characterized yeast species as host/vector systems is severely hampered by the lack of knowledge about transformation conditions and suitable means for stably introducing foreign DNA into the host cell. In addition, auxotrophic mutants are often not available, precluding a direct selection for transformants by auxotrophic complementation. If recombinant DNA technology is to fully sustain its promise, new host/vector systems must be devised which facilitate the manipulation of DNA as well as optimize the expression of inserted DNA sequences so that the desired polypeptide products can be prepared under controlled conditions and in high yield.

Objects of the Invention An object of the invention is a DNA fragment encoding the AOX2 gene of Pichia pastoris wherein said AOX2 gene has a restriction map as shown in Fig 16B.

Brief Description of the Figures Figure 1 is a restriction map of plasmid pYMIl.

Figure 2 illustrates the insertion of a portion of plasmid pYMI1 into the HIS4 locus of the Pichia chromosome.

Figure 3 is a restriction map of plasmid pYJ8.

Figure 4 is a restriction map of plasmid pYMI3a.

Figure 5 illustrates the insertion of a portion of plasmid pYMI3a into the HIS4 locus of the Pichia chromosome.

Figure 6 is a restriction map of plasmid pBPGl-1.

Figure '7 is a restriction map of plasmid pYMI7.

Figure 8 illustrates the insertion of a portion of plasmid pYMI7 into the alcohol oxidase locus of the Pichia chromosome.

Figure 9 illustrates the construction of plasmid pYM39 from plasmids pSAOH5 and pTHBS3.

Figure 10 illustrates the construction of plasmid pYMI6 from plasmids pYM39 and pPG3.2.

Figure 11 illustrates the construction of plasmid pBSAGI5I from plasmid pYMI6 and pBSAG5I.

Figure 12 is a restriction map showing greater detail of plasmid pBSAGI5I than set forth in Figure 11.

Figure 13 illustrates the insertion of a portion of plasmid pBSAGI5I into the alcohol oxidase locus of the Pichia chromosome.

Figure 14 is a restriction map of plasmid pYMll2a.

Figure 15 illustrates the insertion of a portion of plasmid pYMIl2a into the locus of the second Pichia alcohol oxidase gene (AOX2).

Figure 16 is a restriction map of the Pichia inserts in the pBR322—based plasmids pPG4.0 and pPG3.0. The insert shown in Figure 16a is from the locus of the first Pichia alchol oxidase gene (AOXl), while the insert shown in Figure 16b is from the locus of the second Pichia alcohol (AOX2). oxidase (AOX) encoding portion of the AOXl gene locus (with oxidase gene Figure 16c shows the known alcohol reference to Figure 16a).

Figure 17 is a restriction map of plasmid pYM25.

Figure 18 is a restriction map of plasmid pT76H3. throughout represent the restriction enzymes The following abbreviations are used this employed: specification to In the attached figures, restriction sites employed for the manipulation of DNA fragments, but which are destroyed upon ligation, are indicated by enclosing the abbreviation for the destroyed site in parenthesis.

Detailed Description of the Invention In accordance with the present invention, there is provided a process for the site-selective genomic modification of yeasts of the genus Pichia at a predetermined genomic site which comprises transforming a host strain of the genus Pichia with a serially arranged linear DNA fragment a selectable The first and second insertable DNA fragments are each at least about comprising a first insertable DNA fragment, marker gene, and a second insertable DNA fragment. nucleotides in length and have nucleotide sequences which are homologous with separate portions of the native Pichia genome at the site at which the genomic modification is to occur. The selectable marker gene is a gene whose product confers a selectable phenotype upon cells which receive the gene, e.g., gene which allows the cell to synthesize a nutrient required for growth.

DNA vector, DNA to grow under essential that the between the first and second insertable DNA fragments, and an antibiotic resistance gene or a biosynthetic The selectable marker gene, when present on a allows only those cells which receive the vector selective growth conditions. It. is selectable marker gene be positioned that the insertable DNA fragments be positioned in the same orientation with respect to each other as they exist in the genome of the host cell undergoing genomic modification by the linear DNA fragment.

Further in accordance with the present invention, there is provided a serially arranged linear DNA fragment which comprises a first insertable DNA fragment, a selectable The first and second insertable DNA fragments are each at least about marker gene and a second insertable DNA fragment. 200 nucleotides in length, have nucleotide sequences which are homologous with portions of the genomic DNA of species of the genus Pichia, and are oriented with respect to one another in the linear fragment as they exist in the genome of Pichia. The marker gene is positioned between the first and second insertable DNA fragments.

The basic element with which species of the genus Pichia are transformed in accordance with the present invention contains a minimum of three components: a first insertable DNA fragment, a second insertable DNA fragment, and a selectable marker gene.

The first and insertable DNA fragments should each be at least about 200 nucleotides in length, with lengths generally‘ in the range of about 200 up to 5,000 second nucleotides commonly being employed. Preferably, for ease of manipulation and handling, fragments in the range of about 500 up to 2000 nucleotides are employed.

Nucleotide sequences useful as the first and second insertable DNA fragments are nucleotide sequences which are with of the site at which genomic modification is to occur.

Thus, for example, if genomic modification is to occur at the locus of the alcohol oxidase gene, the first and second insertable DNA will be sequences homologous with separate portions of the alcohol oxidase gene locus. homologous separate portions native Pichia genomic fragments employed in accordance with the the two insertable DNA fragments must be oriented with respect to one another in the linear fragment in the same relative orientation as they exist in the Pichia genome.

For genomic modification present invention to occur, The three minimum components of the transforming DNA employed in the practice of the present invention are serially arranged to form a linear DNA fragment wherein the first fragment. selectable marker gene is and the positioned between the insertable fragment second insertable Exemplary selectable marker genes limited to, the ARG4 Saccharomgces cerevisiae, include, but are not gene from Pichia pastoris and the HIS4 gene from Pichia pastoris and S. cerevisiae, the G418 phosphotransferase gene from the E. coli transposable element Tn60l, and the like. Those of skill in the art also recognize that numerous suitable flanking sequences, i.e., the first and second insertable DNA fragments, can be derived from genes which have been isolated front the Pichia pastoris genome. Exemplary genes include, but are not limited to the alcohol oxidase genes (AOXl and AOX2; alcohol dihydroxyacetone synthase gene (DAS), Pichia has two oxidase genes), the the argininosuccinate lyase gene (ARG4), the histidinol dehydrogenase gene (HIS4), and the like.

The transforming linear DNA fragments can include a other DNA heterologous genes, variety of sequences, such as for example, i.e., any gene or portion of a gene not normally found at that locus of the genome where insertion in the host cell is to occur, expression of which is desired in P. pastoris. Generally, the term heterologous refers to DNA not native to the host Pichia cell. The heterologous gene can optionally be combined with a regulatory region which will independently control the production of the heterologous gene product, or the heterologous gene can be expressed in the transformed cell under the influence of the native regulatory region of the gene which has been disrupted in the transformation process.

In addition, the transforming linear DNA fragment employed in the practice of the present invention can also include bacterial plasmid DNA, such as for example, pBR322 or pBR325 sequences. Such bacterial sequences are useful for the in Vitro manipulation and production by amplification in E. coli of these DNA sequences.

An especially useful form for the transforming, linear DNA is as a closed circular plasmid comprising: a first insertable DNA fragment, a second insertable DNA fragment, a selectable marker gene, and bacterial plasmid DNA.

This plasmid can also contain additional DNA sequences as described hereinabove.

In a preferred embodiment, the closed circular plasmid is constructed composed of two portions, the "transforming" portion and the "bacterial" portion. The transforming portion comprises, serially arranged, the first insertable DNA fragment, the selectable marker gene, and the second insertable DNA fragment, wherein the first and second insertable DNA fragments are oriented with respect to one another as they exist in the Pichia genome, with the selectable marker gene positioned between the first insertable DNA fragment and the second insertable DNA fragment. The bacterial portion is then positioned so as to connect the first insertable DNA fragment and the second insertable DNA fragment, thereby forming a closed, circular vector.

The closed, in the previous paragraph can be employed to produce large then and digested with appropriate restriction enzymes to cleave the from the transforming portion of yeast DNA can circular vector prepared as described quantities of plasmid in E. coli, which plasmid is isolated, transforming portion bacterial portion. The linear, then be employed to transform strains of the genus Pichia in order to effect the desired genomic modification.

Of course, the art that the circular plasmid described above can contain additional DNA it is recognized by those of skill in "transforming" portion of the closed sequences. For example, the bacterial sequences employed in of the DNA by amplification in E. coli can be part of the transforming DNA, like the selectable marker Vitro for manipulation and production i.e., the bacterial sequences can, gene sequences, also be positioned between the first insertable DNA fragment and the second DNA fragment. when such a configuration of DNA components is employed, the bacterial sequences would also be incorporated into the genome of the host yeast which is subjected to the process for genomic modification of the present invention.

The transformation of Pichia pastoris has been previously described in copending 666,579 of Stroman et a1., Company. The application Serial No. assigned to Phillips Petroleum experimental procedures employed for the transformation of Pichia pastoris are presented in greater detail below (see Example I). Yeast strains of the genus Pichia can be transformed by enzymatic digestion of the cell walls to give spheroplasts; the spheroplasts are then mixed with the transforming DNA and incubated in the presence of then regenerated in calcium ions and polyethylene glycol, selective growth medium. The transforming DNA includes a selectable marker gene, which allows selection for cells which have taken up transforming DNA, since only transformed cells are capable of survival and growth under the selective growth conditions employed (the selective growth conditions are a function of the selectable marker gene employed as part of the transforming DNA). when strains of Pichia in which the primary alcohol oxidase gene (AOXl) was disrupted were employed as hosts for the expression of heterologous genes, it, was surprisingly observed that the level of" expression of the heterologous gene products, when under the control of some promoters (e.g., AOXl or DAS ‘promoters), relative to the level of expression obtained when a fully was increased several-fold alcohol oxidase-competent host was employed. As a result of this observation and further exploration of this phenomenon, it has been determined that a general method to increase the expression level in host organisms of heterologous which host nutritionally limiting conditions on a substrate for which a genes exists, comprises growing the strain under strong substrate—responsive promoter region exists, wherein the heterologous gene is under the regulatory control of this strong, substrate—responsive promoter.

The "nutritionally limiting conditions" required for the increased gene expression of the present invention can be provided either by feeding the cells limiting amounts of a nutrient or by employing a mutant host which, as a result of the mutation, is nutritionally limited under Thus, desired. to enhance the level of expression of heterologous certain growth conditions. for example, when it is genes maintained under the control of the strong alcohol oxidase or dihydroxyacetone synthase promoters, both of which promoters are responsive to the presence of methanol in the growth media, either the use of a host which is partially defective in its ability to utilize methanol, or the use of with a fully alcohol provide the required that methanol-limited growth conditions host, will nutritionally limiting conditions so oxidase competent enhanced gene expression will be achieved.

It is believed that the method for enhancing the expression of heterologous gene products described herein is a general method useful in any organism for which promoters Thus, by placing a heterologous gene under the control of such a which respond to nutritional limitations exist. promoter region, then culturing the host organism under conditions of nutritional limitation with respect to the nutrient(s) which cause the strong promoter to be turned on, should nutritionally limited growth conditions is to employ" a mutant host organism which is ability to metabolize the nutrient(s) which causes some promoters to be expressed at increased gene expression occur. The presently preferred means to provide partially defective in the much higher levels than in the non—mutant host. In Pichia, this has been demonstrated as described in greater detail in Example V. when a in which the primary alcohol oxidase gene was disrupted by the inventive strain of Pichia pastoris method for genomic modification was cultured with methanol as Carbon source, it was surprisingly found that such strains defective in the primary alcohol oxidase gene were still able albeit at a reduced rate relative to This second to grow on methanol, observation indicated the alcohol methanol utilizing strains of Pichia. wild type Pichia cells. strain has been deposited with the Northern Regional Research site). and is carried by the E. coli Center of’ the United States Department of Agriculture in Peoria, Illinois to insure access to the public upon issuance of this application as a patent and has been assigned the assession number NRRL B—l8022.

A restriction map of pPG3.0 is set forth in Fig. 16b, primary alcohol oxidase gene, where it is compared wiun a genomic fragment of the pPG4.0. The latter fragment has been previously disclosed and described in detail in copending application serial number 666,391. of Stroman et a1., assigned to Phillips Petroleum Company. A comparison of the two alcohol oxidase genes (see Figure 16) makes it clear that the two fragments are not merely overlapping portions of There the same genomic locus. is clearly some homology between the two fragments, as evidenced by the existence of several common restriction sites on the two fragments. The restriction sites common to the two genes, AOXl and AOX2, are denoted in Figure 16 by astericks. However, the existence of several restriction sites on each fragment which. are not on the other that there are differences in the fragments at the nucleotide level. present indicate several The invention will now be described in greater detail by reference to the following non-limiting examples.

EXAMPLES The buffers and solutions employed in the following examples have the compositions given below: lg Tris buffer l2l.l g Tris base in 800 mL of H20; adjust pH to the desired value by (35%) allow solution to cool to room adding concentrated aqueous HC1; temperature before final pH adjustment; dilute to a final volume of 1L.

TE buffer 1.0 mM EDTA in 0.01 g (pH 7.4) Tris buffer LB (Luria-Bertani) 5 g Bacto-tryptone g Bacto—yeast extract 2.5 g NaCl in l L of water, adjusted to pH 7.5 with NaOH Medium B Medium YPD Medium SD Medium SCE Buffer PEG Solution .2% NH4PO4 .2% Na2HPO4 .o13% MgSO4-7H2O .o74% CaCl2-2H2O pg/mL biotin ——‘f\)O©|-‘CD pg/mL thiamine pg/mL tryptophan 0.4% dextrose .2% casamino acids % Bacto-yeast extract 2% Bacto-peptone 2% Dextrose .75 g yeast nitrogen base without amino acids (DIFCO) 2% Dextrose in 1 L of water Q Sorbitol my EDTA 50 mg DTT .1 g Sorbitol .47 g Sodium citrate 0.168 g EDTA mL H20 --pH to 5.8 with HCl E Sorbitol mg CaCl2 --filter sterilize % polyethylene glycol-3350 10mg CaCl2 lomg Tris-HCl (pH 7.4) --filter sterilize SOS 1 Q Sorbitol 0.3x YPD medium m1~_a CaCl2 The following abbreviations are used throughout the examples with the following meaning: EDTA ethylenediamine tetraacetic acid SDS sodium dodecyl sulfate DTT dithiothreitol EXAMPLE I Pichia_pastoris Transformation Procedure A. Cell Growth . Inoculate a colony of Pichia pastoris GS115 (NRRL Y-15851) into about 10 mL of Y?D medium and shake culture at °C for 12-20 hrs.

. After about 12-20 hrs., dilute cells to an ODSOO of about 0.01-0.1 and maintain cells in log growth phase in YPD medium at 30°C for about 6-8 hrs.

. After about 6-8 hrs, inoculate 100 mL of YPD medium with 0.5 mL of the seed culture at an ODSOO of about 0.1 (or Shake at 30°C for about 12-20 hrs.

. Harvest culture when ODSOO is about 0.2-0.3 (after approximately 16-20 hrs) by centrifugation at 1500 g for 5 equivalent amount). minutes.

B. Preparation of Spheroplasts . wash cells once in 10 mL of sterile water. (All centrifugations for steps 1-5 are at 1500 g for 5 minutes.) 2. Wash cells once in 10 mL of freshly prepared SED.

. Wash cells twice in 10 mL of sterile 1 Q Sorbitol.

. Resuspend cells in 10 mL SCE buffer.

. Add 5-10 pL of 4 mg/mL Zymolyase 60,000 (Miles Laboratories). Incubate cells at 30°C for about 30-60 minutes.

Since the preparation of spheroplasts is a critical step in the transformation procedure, one should monitor spheroplast. formation as follows: add 100 uL aliquots of cells to 900 uL of 5% SDS and 900 pL of 1 N Sorbitol before or just after the addition of zymolyase and at various times Stop the incubation at the point where cells lyse in SDS but not in sorbitol (usually between 30 and 60 minutes of incubation). during’ the incubation period.

. Wash spheroplasts twice in 10 mL of sterile 1 lg Sorbitol by centrifugation at 1000 g for 5-10 minutes. (The time and speed for centrifugation may vary; centrifuge enough to pellet spheroplasts but not so much that they rupture from the force.) . wash cells once in 10 mL of sterile Cas.

. Resuspend cells in total of 0.6 mL of Cas.

C. Transformation . Add DNA samples (up to 20 pL volume) to 12 X 75 mm (DNA should be in water or TE for Inaximum transformation frequencies with small it is advisable to add about 1 pL of 5 mg/mL sonicated E. coli DNA to each sample.) . Add 100 pL of spheroplasts to each DNA sample and incubate at room temperature for about 20 minutes.

. Add 1 mL of PEG solution to each sample and incubate at room temperature for about 15 minutes. sterile polypropylene tubes. buffer; amounts of DNA, . Centrifuge samples at 1000 g for 5-10 minutes and decant PEG solution.

. Resuspend samples in 150 uL of SOS and incubate for minutes at room temperature.

. Add 850 pL of l N Sorbitol and plate aliquots of samples as described below. sterile D. Regeneration of spheroplasts- l. Recipe for Regeneration Agar Medium: a.‘ Agar-KCl— 9 g Bacto—agar, 13.4 g KCl, 240 mL H20, autoclave. b. 10X Glucose— 20 g Dextrose, 100 mL H20, autoclave. c. 10X SC- lOO mL H20, (Add any desired amino acid or nucleic acid up ‘to a concentration of 200 pg/mL before or after autoclaving.) d. Add 30 mL of 10X Glucose and 30 mL of 10X SC to 240 mL of the melted Agar-Kcl solution. Add 0.6 mL of 0.2 mg/mL biotin and any other desired amino acid or nucleic acid to a concentration of 20 pg/mL. 55—60°C.

. Plating of Transformation Samples: .75 g Yeast Nitrogen Base without amino acids, autoclave.

Hold melted Regeneration Agar at Pour bottom agar layer of 10 mL Regeneration Agar per plate at least 30 minutes before transformation samples are ready. Distribute 10 mL aliquots of Regeneration Agar to tubes in a 45-50°C bath during the period that transformation samples are in SOS. Add aliquots of transformation samples described above to tubes with Regeneration Agar and pour onto bottom agar layer of plates.

. Determination of Quality of Spheroplast Preparation: Remove 10 pL of one sample and dilute 100 times by addition to 990 pL of 1 Q Sorbitol. Remove 10 uL of the 100 fold dilution and dilute an additional 100 times by addition to a second 990 pL aliquot of 1 E Sorbitol. Spread 100 pL of both dilutions containing YPD medium to determine the concentration of unspheroplasted whole cells Add 100 pL of each dilution to ug/mL spheroplasts. on agar plates remaining in the preparation. mL histidine to of Regeneration Agar supplemented with 40 total Good values for‘ a transformation experiment are 1-3 X 107 total regeneratable spheroplasts/mL and about 1 X 103 whole cells/mL.

. Incubate plates at 30° C for 3-5 days. determine regeneratable Example II Site-specific Insertion of the Saccharomyces ARG4 Gene and pBR322 and Deletion of the Pichia HIS4 in GSl9O (NRRL Y—l80l4} FIGURE 1 shows plasmid pYMIl and Figure 2 shows a diagram of events which result in the plasmid‘s site-directed insertion into the P. pastoris genome. The vector is designed to insert the Saccharomgces ARG4 gene and DNA sequences from pBR322 into the Pichia HIS4 locus, simultaneously deleting the entire HIS4 gene locus from the Pichia Other could easily be genome. sequences, such as expression cassettes, inserted into pYMIl and then similarly incorporated into the E; pastoris genome. fragments can be obtained from plasmid pYJ8 (available in an in situ, Pichia Illinois, see Figure 3), As a a 2.6 kbp (available in an E. selectable marker, the pYMIl vector contains HindIII-Sall fragment from pYM25 host as NRRL B-18015; argininosuccinate coli which encodes the see Figure 17) Saccharomgces lyase gene HIS4 gene-flanking fragments at its termini.

The g; pastoris arg4 strain, GSl9O (NRRL Y-18014), was transformed with EcoRI—cut pYMIl to arginine prototrophy (Ar9+). 40 pg/mL of histidine to avoid exerting selection pressure The regeneration agar medium was supplemented with against transformants which required histidine as a result of the HIS4 gene deletion.

V The Arg+ transformants were then screened for those as a result of deletion of the HIS4 To screen for His- which had become His‘ gene. transformants, the regeneration agar with the embedded Arg+ colonies was transferred to a sterile 50 mL tube which contained 25 mL of sterile water.

The agar was then pulverized by mixing with a Brinkman Instruments Polytron homogenizer' at setting 5 for about 1 minute. The yeast cells were separated from the agar by filtration through three layers of sterile gauze. A portion of the yeast cells was then sonicated, diluted and spread on SD medium agar plates supplemented with 40 pg/mL of histidine.

For sonication, samples of cells were diluted to an A600=0.l and sonicated for 10 seconds with a Sonifier Cell Disrupter 350 (Branson Sonic Power Co.) at power setting 4, a treatment which is sufficient to separate cells but not to After 2-3 days, on the SD plus histidine agar plates were replica plated to reduce cell viability. colonies which grew sets of SD plates, one with and one without histidine.

The proportion of Arg+ His‘ colonies averaged 0.7% Genomic DNA from three Arg+ examined by Southern blot hybridization. of the total Arg+ transformants.

His‘ strains was The entire HIS4 gene was absent in all three and the linear plasmid was inserted as shown at the bottom of FIGURE 2.

Besides the fact that the GSl90—pYMIl strain requires histidine and no longer requires arginine for growth, no other changes in nutritional requirements or growth rates were observed.

EXAMPLE III Deletion of the HIS4 Gene from P. pastoris NRRL Y-11430 FIGURE 4 shows plasmid pYMI3a and Figure 5 sets forth the events which result in the linear insertion of a fragment from the plasmid into the HIS4 locus of 3; pastoris strain NRRL Y-11430. gene (G4l8R) from pBPGl-1 (available from the United States The vector contains the G418 resistance Northern Regional Research Center strain NRRL The G4l8R Department of Agriculture, in Peoria, Illinois, in an E. coli host as B-18020; see Figure 6) as the selectable marker.

(PARS1 site) fragment and inserted into of pYJ8 (NRRL B—l5889; see 2.7 kbp gglll Pichia HIS4 gene. site Figure 3), Transformants were selection, . Recipe for regeneration agar medium: a. Agar-sorbitol- 9 g bacto-agar, 54.6 g Sorbitol, 240 mL H20, autoclave. b. lOx glucose— 20 g Dextrose, 100 mL H20, autoclave. c. 10x YP— 10 g yeast extract, mL H20, d. Add 30 mL of 10x glucose and 30 mL of 10x YP to 240 mm; of melted agar-sorbitol solution.

Hold melted 55°C-60°C.

. Plating of transformants. g peptone, 100 autoclave. regeneration medium agar at Sample: At least 30 minutes before transformation samples were ready, 10 mL/plate bottom agar layer of regeneration medium agar supplemented with 600 pg/mL of G418 was poured.

During the period the transformation samples were in SOS, 10 mL aliquots of regeneration medium agar (without G418) were distributed to tubes in a 45-50°C bath. when transformation samples were ready, aliquots of the samples were added to the tubes with the regeneration agar and poured onto the 10 mL bottom agar layer containing G418. Plates were incubated at °C for 3-5 days.

After colonies had formed on the regeneration agar plates with G418, the cells were screened for their ability to grow without histidine. Cells were extracted from the regeneration agar, sonicated, and spread on SD medium agar plates supplemented with 40 pg/mL of histidine as described After 2-3 days incubation at 30°C, the colonies were replica plated onto SD medium agar plates with and without histidine.

Approximately 0.1% of the G4l8R (2 out of approximately 2,000 in Example II. colonies were His" Southern blot showed that the HIS4 gene was deleted from the genomes of both His‘ strains and that both genomes contained the G4l8R gene as shown in Figure 5. One screened). hybridization experiments of these His‘ strains, given the laboratory designation KM31 (available from the United States Department of Agriculture, Illinois, as NRRL Y-18018) has been successfully transformed with several HIS4-containing Pichia-based plasmids such as, pSAOH5 (available in an E. coli host as NRRL B-15862), which provides further evidence that KM3l is specifically a EIS4 Northern Regional Research Center in Peoria, for example, gene deletion organism.

This is the first time that "wild type" E; pastoris NRRL Y-ll43O has been transformed directly (i.e., without first isolating and characterizing an auxotrophic derivative). A possible advantage of these HIS4 mutant strains is that since they were constructed by the site-specific insertion/deletion method, they are free of secondary mutations which probably exist in Pichia auxotrophic hosts which are produced, for example, by chemical mutagenesis, such as for example GSll5 ,(NRRL Y-15851) and GSl9O (NRRL Y-18014). for example pSAOH5 (see Figure 9) are primarily composed of sequences from pBR322 and the Pichia HIS4 gene, these autonomous vectors have little homology with the genome of these Pichia HIS4 deletion hosts, frequently integrate. and, therefore, should not Example IV Disruption of the Primary Alcohol Oxidase Gene Pichia strains lacking the alcohol oxidase genes (the primary alcohol oxidase gene is referred to herein as AOXI and the secondary alcohol oxidase gene is referred to herein as AOX2) are of interest for at least two reasons. in the expression by methanol.

First, as an aid studies on regulation of gene For example, with a mutant strain defective in the AOXI and AOX2 genes, as described in greater detail in Example VII, evidence can be obtained as to whether methanol or some other metabolite (formaldehyde, formate, etc.) is the actual inducing molecule of methanol regulated genes. A second interest in an AOX—defective Pichia strain lies lJ1 the possibility that such a strain might express higher levels of heterologous gene products as described in greater detail in Examples V and VI.

To disrupt the AO gene, (FIGURE 7). The plasmid was constructed by inserting a 2.9 kbp gamfil-ggll fragment from plasmid pYM25 (NRRL B-18015; see Figure 17) which contains the Saccharomyces ARG4 gene into ggmfil-cut pPG4.0 (NRRL B-15868; restriction map of the Pichia portion of this plasmid). The plasmid pYMI7 was created see Figure 16a for a Colonies which resulted were then replica plated onto a set .1% glucose (instead of SD medium agar plates (containing histidine) with. the following carbon sources: 1) no carbon; 2) 0.5% methanol; and 3) 2% glucose. About 81.0% of the Arg+ colonies could not grow normally on methanol. Southern blot analysis of genomic DNA from two of these methanol nonutilizers, i.e., KM71 and KM72, that the AOXl gene was disrupted in these strains, and that the vector was inserted as shown at the bottom of Figure 8.

The PPFl—pYMl7 constructions having the genotype: confirmed alcohol oxidase defective (his4 aoxl::SARG4) are of great potential value. For example, since the strain is his4, Pichia which contain the HIS4 gene as a selectable pSAOH5 (NRRL B-15862; see Figure 9), can be transformed into this host, as vectors marker, such as, for example, described more fully in Example V, below.

Example V Methanol-Regulated Expression of the lacz Gene in an Alcohol Oxidase-Defective Mutant Strain of Pichia pastoris This describes expression of the Zacz gene in the Pichia host KM7l (a example experiments on the PPFl-pYMI7 alcohol oxidase defective construction) which was prepared as described in Example IV.

The Aoxl' was KM7l (his4 aoxl::SARG4) and the Aoxl* host was PPFl (arg4 his4; NRRL Y—l80l7). The AOXl promoter—JacZ cassette transformed into both strains was on the plasmid pSAOH5 NRRL B-15862). Several stable His* transformants from both strains were isolated. host for these comparative experiments expression (see Figure 9, Their genomic DNAs were examined by Southern. blot analysis to obtain a matched set of strains, each containing pSAOH5 integrated at the AOXl promoter locus. Similarly, the dihydroxyacetone synthase (DAS) promoter-lacz gene fusion was transformed into KM7l and PPFl on plasmid pT76H3 (see Figure 18; NRRL B—l8000) by selecting for histidine prototrophy. _ Each of the four strains was initially grown in SD medium except with 2% glycerol as sole carbon and energy source instead of 2% glucose. Each culture was then shifted, i.e., collected by centrifugation and transferred to a different medium, i.e., SD medium with 0.5% methanol as sole carbon source. Samples of these cultures were assayed for B—galactosidase, with the results summarized in Table I.

Table I B-Galactosidase, units/pg* *5-Galactosidase Assay A. Solutions required: 1. Z-buffer: Final concentration Na2HP04 . 7H20 16.1 g 0.06 1~_a NaH2PO" 5.5 g 0.04. y1_ KCl 0.75 g 0.01 91 Mgso4 . 71120 0.246 g 0.001 13 2—mercaptoethano1 2.7 mL 0.05 g fill up to 1L; pH should be 7 . O-Nitrophenyl—B—D—galactoside (ONPG) Dissolve 400 mg ONPG (Sigma N—ll27) in 100 mL of distilled water to make a 4 mg/mL ONPG solution.

B. Assay Procedure: 1. Withdraw an aliquot from the culture medium (20-50 ODGOO of yeast cells), centrifuge and wash cell pellet with cold sterile water.

. Add 1 pL of 40% 2 buffer to the cell pellet and 0.2 pg of acid washed 0.45-0.50 mm glass beads. on ice.

Hold all samples Vortex the mixture at the highest setting four (4) times for one minute each time. Samples should be held on ice for at least one minute between vortexings.

. Transfer lysates to tubes and Transfer the and hold. the microcentrifuge centrifuge in a microfuge at 4°C for 5 minutes. supernatants to fresh microcentrifuge tubes extracts on ice.

. The concentration of total protein in an extract was estimated using the Bio-Rad Laboratories (Bradford) protein assay method. For this the Bio-Rad Dye Reagent Concentrate was diluted with four volumes of deionized H20 and filtered through Whatman 3M paper. was then prepared by adding 3, 10, 30, and 100 pg of bovine serum albumen (BSA) in 100 pL Z buffer to a set of 13 x 100 mm glass tubes each of which contained 2.5 mL of the dye A standard concentration curve reagent. The samples were mixed and held at room temperature for 5 minutes and their optical densities at 595 nm determined. For the extracts, 3, l0, and 30 pL samples were brought to 100 pL with a solution containing the Z buffer and assayed. for protein content as described above. A protein concentration value for each extract was then interpolated using the BSA concentration curve.

. For the B-galactosidase 10 pL of a 10x dilution of extract was added to mL of 2 buffer and the mixture was incubated for 5 minutes at 30°C.

. Start reaction by adding 0.2 mL of ONPG (4 mg/ml), vortex.

. Stop reaction by adding 0.5 ml of a 1 lg Na2CO3 solution at appropriate time points (usually between 1 and 30 assays, minutes, and at A420 <1).

. Read absorbance of supernatant at 420 nm.

C. Calculation of B-galactosidase Activity Units: U = l nmole of orthonitrophenol (ONP) minute at 30°C and a pH 7. nmole of ONP has an absorbance at 420 nm (A420) of 0.0045 with a 1. cm pathlength; thus, an absorbance of 1 at 420 nm represents 222 nmole ONP/mL, or 378 nmole/1.7 mL since the total volume of supernatant being analyzed is 1.7 mL.

Hence, Units expressed in the Tables are calculated: formed per U : A420 X 378 t(min) Each of the four cultures showed almost no detectable B—galactosidase activity during the glycerol-growth phase. About 10-20 hours after shifting to methanol medium, the two cultures which contained the AOX1-Zacz and DAS—1acZ expression cassettes in the Aox1* host showed B-galactosidase activity leveling off at about 20 units of B—galactosidase activity per pg the AOXl—1acZ cassette showed activity reaching around 60 units/pg. of protein. in the Aox1' background The DAS—ZacZ However, cassette in the Aoxl- host showed an increase in B—galactosidase activity levels as well. Thus, the transformed Aoxl- host, KM7l, expressed B—galactosidase at -3 times the level of the transformed isogenic—Aoxl* strain, PPF1.

Example VI Insertion of the Hepatitis B Surface Antigen Gene and Deletion of the AOXl Gene In this the entire coding sequence of the AOX1 example of site-directed insertion/deletion, gene was deleted and the Hepatitis B surface antigen (HBsAg) gene was inserted under control of the AOXI gene promoter For this 2; pastoris host plasmid pBSAGI5I was created (deposited in an which remains in the genome. construction, E. coli host with the Northern Regional Research Center of the United States Department of Agriculture, in Peoria, Illinois, and available to the public without restriction upon issuance of a patent from this application, with accession number NRRL B-18021; Figure 12). The plasmid contains a 1.0 kbp fragment from sequences flanking the '—terminus of the AOX1 gene followed tar the hepatitis B (HBsAg) and the 300 bp AOXl terminator fragment, all assembled as shown in Figures 9, 10 and 11. The expression cassette was followed by the 2.7 kbp fragment encoding the Pichia HIS4 gene and finally, a 1.5 kbp B3311 fragment containing sequences 3' to the AOX1 gene. when pBSAGI5I was digested with gglll, a 7.2 kbp linear vector was released which contained 0.85 kbp of 5’-AOX1 gene surface antigen sequence sequence at one terminus and 1.1 kbp of 3'-AOXI sequence at BglII-cut pBSAGI5I was transformed into GSll5 by selecting for histidine prototrophy, the other terminus. transformants were extracted from the regeneration agar and sonicated as described in Example II, and spread on SD medium agar plates with 0.1% glucose (instead of 2.0%). resulted were then replica plated onto minimal agar plates The colonies which with the following carbon sources: 1) no carbon source, 2) 0.5% methanol, On the average 32% of the colonies examined could not grow normally on methanol. and 3) 2% glucose.

Southern blot analysis of the genomic DNAs from two of the methanol nonutilizers demonstrated that the AOXl gene was deleted and that the vector sequences were inserted as shown in Figure 13. when grown in methanol, the GSll5-pBSAGI5I strain (aoxlz :1-IBsAg-HIS4) than expressed by fully alcohol oxidase competent cells similarly expressed HBsAg in higher levels transformed.

Example VII Identification of the Second Alcohol Oxidase Gene of P. pastoris by the Site-Directed Insertion Technique The presence of a second alcohol oxidase gene can ) Southern blots in which probes from either AOX CDNA or a genomic DNA be inferred from the following’ observations: were hybridized to restricted Pichia genomic DNAs always showed at least two bands; 2) two Pichia genomic DNA fragments were originally isolated which were similar but not identical to each other (see Figure 16); and 3) mutant Pichia strains such as KM71 and GS115-pBSAGI5I in which the primary AOX gene (AOX1) was deleted or disrupted could still grow on The growth rate and AOX activity in methanol—growth cells of these Aox” much less methanol and contained alcohol oxidase activity. strains.

(AOX2) is expressed at a lower level or that its product is less active it was demonstrated that the Pichia DNA fragment in pPG4.0 contains the AOXl gene. strains was than in isogenic-Aoxl* Therefore, it appears that the second AOX gene on methanol. In Example IV, The most convincing method of demonstrating that the genomic DNA fragment fronx pPG3.0 contains at least a portion of the AOX2 gene is by constructing a mutant strain in which this putative AOX2 gene has been disrupted or deleted. For this, constructed (Figure 14). insertion Comparison of the This linear vector was transformed into the Aoxl‘ KM7l, (aoxl his4::SARG4), isolated by selecting for Strain, and transformants were histidine prototrophs. The transformants were then screened for the ability to utilize methanol by replica plating onto sets of agar plates.

The untransformed Aoxl' strain KM71 grew so slowly on methanol plates that if methanol was included in the agar, it evaporated before significant growth could be observed.

This problem was solved by feeding methanol to the cells in about 0.2 mL of 100% methanol was placed under the lid of a plate which contained The plate was left at room temperature, and the lid was replaced every 2 to 4 days After about l-2 weeks, the difference in methanol growth of wild type (Aoxl* Aox2*), (Aoxl‘ Aox2*) and (Aoxl‘ Aox2‘) was vapor phase. For this procedure, no carbon source in the agar medium. with a fresh methanol—containing lid. and the mutant strains, clear.

Following the vapor-phase feeding procedure, it was found that about 0.1% of His+ strain were unable to grow on methanol. the Aoxl‘ Aox2‘ His* Southern filter hybridization procedure. transformants from the Aoxl’ DNAs from eight of transformants were analyzed by the Three of these DNAs contained the linear pYMIl2a ‘vector inserted as shown in Figure 15. Analysis with one of the Aoxl’ Aox2' double KM7l21 (NRRL Y-18019), showed that the strain absolutely does not grow on methanol and that the strain does not have detectable AOX activity. From these results with KM7l2l, it is clear that the Pichia fragment in pPG3.0 does contain sequences from a second AOX gene and that, other than mutants, these two alcohol oxidases, no other methanol-oxidizing activities exist in P. pastoris.

The examples have been provided merely to illustrate the practice of the invention and should not be read so as to limit the scope of the invention or the appended claims in any way. Reasonable variations and modifications, not departing from the essence and spirit of the invention, are contemplated to be within the scope of patent protection desired and sought.

Claims (1)

1. A DNA fragment encoding the AOX2 gene of Pichia pastoris, wherein said AOX2 gene has the restriction map as shown in FIG. 16b of the