Process for producing polypeptides and for place-specific modification of genom ofpichia yeasts

The present invention relates to a site-specific modification of the genome of Pichia yeast at a predetermined site of the genome, wherein

- replacing the internal region of a DNA fragment homologous to the genomic DNA segments of the yeast strain of the genus Pichia by a DNA fragment containing a selectable marker gene and, optionally, a regulatory region and a heterologous gene, such that all 5'- at least 200 nucleotides at each end and at each 3 'end of a fragment of DNA homologous to the genomic DNA of the yeast strain of the genus Pichia, and

- transforming a yeast strain of the genus Pichia with the above DNA fragment.

The present invention further relates to a method for producing polypeptides in Pichia pastoris AOX 5 host cells.

Description: 30 pages (including 19 pages)

CQ

HU 208 552

HU 208 552 Β

The present invention relates to a method for site-specific modification of the genome of yeast Pichia and to the production of polypeptides.

The present invention relates to the field of recombinant DNA technology. More particularly, the present invention relates to an integrative transformation of yeast, a site-specific mutation of yeast.

Recombinant DNA technology has undergone significant development in recent years, with the controlled production of microorganisms to produce a large number of useful polypeptides. Many eukaryotic polypeptides, such as human growth hormone, leukocyte interferon, human insulin and human proinsulin, can be prepared from several microorganisms. As technology continues to evolve, it will be possible for microorganisms to produce many more useful polypeptides in the future. A common element often used in recombinant technology is the plasmid, which is an extrachromosomal double-stranded DNA that is found in many microorganisms. In naturally occurring microorganisms, the plasmid is usually found in multiple copies per cell. In addition to natural plasmids, many artificial plasmids and hybrid vectors have been produced. However, the host cell is not always capable of retaining the plasmid. Instead, the cell loses its plasmid-producing ability and, after a few generations, the plasmid disappears. Thus, the stable incorporation of foreign DNA into a suitable host is an important and important procedure.

Escherichia coli is generally used as a host in recombinant DNA technology used to produce polypeptides, although in many cases E. coli is not suitable as a host cell. For example, E. coli contains a number of toxic pyrogenic factors from which the polypeptide produced for therapeutic purposes must be purified. The degree of purification required will, of course, depend on the particular polypeptide. In addition, E. coli's proteolytic activity limits the yield of several useful products. A further disadvantage is that several heterogeneous gene products produced by E. coli can be recovered in insoluble form. For these and other reasons, interest has turned to alternative hosts. As a result, the use of eukaryotic organisms for the production of polypeptides is increasing.

The production of polypeptides by eukaryotic systems such as yeast has significant advantages over the production of recombinant DNA encoded polypeptides by prokaryotic systems such as E. coli. Yeast has been used for a wide variety of fermentations for centuries compared to recent uses of E. coli fermentations. Yeast is usually cultured to a higher cell density, both bacterial, and readily used for continuous fermentation. U.S. Patent No. 4,414,329 describes the cultivation of yeasts, such as Pichia pastoris, up to a high cell density above 100 g / l. A further advantage of yeasts is that many of the body's vital functions, such as oxidative phosphorylation, are carried out in intracellular organs and thus are not exposed to possible harmful factors caused by production of polypeptides foreign to the original host cell. As a eukaryotic organism, yeast may be able to glycosylate the expressed polypeptide product, which is important in cases where the glycosylation provides the bioactivity of the polypeptide product. It is also possible that the yeast, as a eukaryotic organism, contains the same preferred codes as the higher organisms, which further enhances the efficiency of the production of products derived from mammalian genes or complementary DNA obtained by reverse transcription with mammalian mRNA.

The use of little known yeast varieties as a host / vector system is often hampered by a lack of knowledge of transformation conditions and the difficulty of introducing foreign DNA into a host cell. In addition, auxotrophic mutation is often not possible, which precludes direct selection of the transformed products by auxotrophic complementation. A precondition for the development of recombinant DNA technology is the need to find new host / vector systems that allow for DNA treatment, optimal expression of the inserted DNA sequence, and thus controlled production of the desired polypeptide product in high yield.

According to the present invention, a novel method has been developed for site-specific modification of the genome of the yeast Pichia genus.

If the yeast is transformed with a linear DNA fragment that contains at each end a DNA sequence that is homologous to the host genome, each having at least 200 nucleotides in length and at least one additional DNA fragment, such as a selectable marker, the marker and a side insert DNA sequence. it is picked up frequently by the host. Transformation with linear DNA results in a modified yeast strain in which the DNA sequence is disrupted and / or deleted at the site of insertion and incorporates the selectable marker and any DNA that is part of the transformed linear DNA fag fragment into the host yeast genome.

Foreign DNA inserted into the host yeast genome as described above is stably maintained in the host for several generations. The method of the present invention makes it possible to eliminate the loss of foreign DNA due to plasmid instability when the foreign DNA is part of a self-replicating extrachromosomal fragment or when the foreign DNA is incorporated into the genome as part of the circular DNA molecule. Such circular DNA molecule insertion occurs when a foreign DNA sequence is inserted which is a direct repeat of the host genome sequences. Since such direct repetitive sequences form the basis for further recombination, the foreign DNA inserted therein is not sufficiently stable.

The site-specific modification of the genome according to the present invention allows the production of mutants that lack the

HU 208 552 Β which gene. By eliminating a substantial portion of the gene targeted for the mutation, the probability of reverse mutation is practically reduced to zero. Thus, the strains obtained by the mutation of the invention form a highly stable mutant.

The site-specific modification of the genome according to the invention, together with in vitro recombinant DNA technology known to those skilled in the art, permits arbitrary and precise modification of the host cell genome, e.g., the insertion of one or more foreign genes into the host genome. genes.

Surprisingly, it has been found that methanol-induced expression of foreign genes in strains of the genus Pichia is significantly enhanced by using a Pichia strain in which the primary alcohol oxidase gene is disrupted as a host cell for gene expression.

It has also been found that the Pichia pastoris strain also has a second alcohol oxidase gene, which allows the host strain whose primary alcohol oxidase gene is degraded to continue to grow in methanol, albeit at a reduced rate compared to native Pichia pastoris.

Brief Description of the Drawings:

First Fig. 3A shows the cleavage map of plasmid pYMI1,

Second Figure 4A shows the insertion of a portion of plasmid pYMI1 into the HIS 4 site of the Pichia chromosome,

Third Fig. 4A is a cleavage map of plasmid pYJ8.

4th Fig. 4A is a cleavage map of plasmid pYMI3 A,

5th Figure 4A shows the insertion of a portion of plasmid pYHMI3A into the HIS4 site of the Pichia chromosome,

6th Fig. 3A is a cleavage map of plasmid pBPG1-1;

7th Fig. 4A is a cleavage map of plasmid pYMI7.

8th Fig. 3A shows the insertion of a portion of plasmid pYMI7 ​​into the alcohol oxidase site of the Pichia chromosome,

9th Fig. 3A shows the construction of plasmid pYM39 from pSAOH5 and pTHBS3;

10th Fig. 3A shows the construction of plasmid pYMI6 from plasmids pYM39 and pPG3.2,

11th Fig. 6A shows the construction of plasmid pBSAGI5I from plasmids pYMI and pBSAG5I,

12th all. Figure 5a shows a more detailed cleavage map of plasmid pBSAGI5I than

13th Figure 1A shows the insertion of a portion of plasmid pBSAGI5I into the alcohol oxidase site of the Pichia chromosome,

14th Fig. 4A is a cleavage map of plasmid pXMI12A,

15th Figure 4A shows the insertion of a portion of plasmid pYMI12A into the site of the second Pichia alcohol oxidase gene (A0X2),

16th Fig. 4A is a cleavage map of Pichia inserts in pBG322 based plasmids pPG4.0 and pPG3.0,

The insertion in Figure 16a is from the site of the first Pichia alcohol oxidase gene (A0X1), whereas

The insertion in Figure 16b is from the site of the second Pichia alcohol oxidase gene (AOX2),

Figure 16c contains the coding part of the known alcohol oxidase (AOX) for the AOX1 gene site (with reference to Figure 16a),

17th Fig. 4A is a cleavage map of plasmid pXM25.

18th Figure 1c is a cleavage map of plasmid pT76H3.

19th Figure 3B shows the conversion of the KpnI cleavage site to the BglII site

20th Fig. 6A shows the destruction of the BamHI site by the "filing in" method.

The restriction enzyme used is hereinafter designated by the following abbreviations:

<td>abbreviations</td><td>Restriction enzymes</td>

<td>Dig</td><td>AsuII</td>

<td>B</td><td>Bam</td>

<td>b₂</td><td>BglII</td>

<td>C</td><td>Cla</td>

<td>Ri</td><td>Eco</td>

<td>r₅</td><td>RV</td>

<td>h₃</td><td>Hind</td>

<td>Hpi</td><td>Hpa</td>

<td>kp</td><td>KpnI</td>

<td>nr</td><td>NruI</td>

<td>ps</td><td>Pst</td>

<td>pv₂</td><td>PvuII</td>

<td>Rs</td><td>RsaI</td>

<td>S</td><td>Sáli</td>

<td>s₃</td><td>Sau3AI</td>

<td>sm</td><td>Smal</td>

<td>sp</td><td>SphI</td>

<td>St</td><td>Stu</td>

<td>th</td><td>thai</td>

<td>Xb</td><td>XbaI</td>

<td>xH</td><td>XhoI</td>

Disturbed recognition sites during the ligation and cloning process are indicated in the figures by the above abbreviations in parentheses. For example, "(Rs / Pv₂) "Refers to the Rsal and PvuII enzymes which recognize the GTAC and CAGCGT sequences. Both enzymes cleave the DNA symmetrically, resulting in blunt-ended fragments with the 5 'end being AC and CTG and the 3' end being GT and CAG. These fragments can be cross-ligated to give a mixture of the original recognition sequences (CTCTG and / or CAGAC) which cannot be cleaved with either Rasl or PvuII.

According to the invention, yeasts of the genus Pichia are located at a predetermined site of the genome

Specifically, the Pichia genus is transformed with a sequence of linear DNA fragments comprising a first insertional DNA fragment, a selectable marker gene, and a second insertional DNA fragment. Each of the first and second insertional DNA fragments is at least about 200 nucleotides in length and has a nucleotide sequence that is homologous to a distinct region of the native Pichia genome for modification. The selectable marker gene is a gene that confers a selectable phenotype on the host cell, such as an antibiotic resistance gene or a biosynthetic gene that synthesizes the nutrient required for cell development. If the selectable marker gene is located on a DNA vector, only the cells that have received the vector DNA will grow under the particular developmental conditions. It is important that the selectable marker gene be located between the first and second insert DNA fragments, and that the insert DNA fragments are located in the same orientation relative to each other as in the genome of the host cell to be modified by the linear DNA fragment.

Thus, the basic element used for the transformation of Pichia according to the invention consists of at least three components:

- first insert DNA fragment,

a second insert DNA fragment and

- selectable marker gene.

The first and second insertion DNA fragments are at least about 200 nucleotides in length, generally about 200-5,000 nucleotides in length. For ease of use, fragments of about 500 to 2000 nucleotides in length are preferably used.

The nucleotide sequence of the first and second insert DNA fragments is homologous to a particular portion of the native Pichia genome that is targeted for genomic modification. For example, if the genomic modification is to be carried out at the site of the alcohol oxidase gene, the sequence of the first and second insertion DNA fragments is homologous to a separate region of the alcohol oxidase gene. The genomic modifications of the invention have the orientation of the two inserted DNA fragments within the linear fragment relative to the relative orientation of the Pichia genome.

In accordance with the present invention, the three required components of the DNA used for transformation form a linear DNA fragment, in which the selectable marker gene is located between the first insertion fragment and the second insertion fragment. Non-limiting examples of a selectable marker gene include the ARG4 gene of Pichia pastoris and Saccharomyces cerevisiae, the HIS4 gene of Pichia pastoris and S. cerevisiae, E. the G418 phosphorus transferase gene of the transposable element of TnóO1 coli and other similar genes. One of ordinary skill in the art will recognize that a number of suitable insertion sequences, i.e., first and second insertable DNA fragments, can be deduced from genes that can be isolated from the Pichia pastoris genome. Non-limiting examples of these genes include the alcohol oxidase gene ((AOX1 and AOX2), Pichia contains two alcohol oxidase genes), the dihydroxyacetone synthase gene (DA), the arginine succinase lyase gene (ARG4), the histidinol dihydrogenase gene (HIS4). ) and other similar genes.

The linear DNA fragment used for the transformation may also contain a number of other DNA sequences, such as heterologous genes, i.e. genes or gene fragments that are not usually found at the site of host gene insertion expressed in P. pastoris. A heterologous term generally refers to DNA that is not found in the Pichia host cell. The heterologous gene may optionally be combined with a regulatory region that independently regulates the production of the heterologous gene product, or the heterologous gene may be expressed in the transformed cell by the native regulatory region of the gene disrupted during transformation.

According to the invention, the linear DNA fragment used for transformation may also contain bacterial plasmid DNA, such as pBR322 or pBR325 sequences. These bacterial sequences are used for the in vitro manipulation and production of DNA sequences in E. coli.

Particularly preferred for transformation is linear DNA introduced into a closed circular plasmid comprising a first insertional DNA fragment, a second insertional DNA fragment, a selectable marker gene and bacterial plasmid DNA. This plasmid may also contain additional DNA sequences as described above.

Preferably, the closed circular plasmid consists of two parts, the "transforming" part and the "bacterial" part. The transforming portion comprises a sequenced first insert DNA fragment, a selectable marker gene and a second insert DNA fragment, wherein the first and second insert DNA fragments have the same orientation relative to each other, the selectable marker gene is the first insert DNA fragment and the second insert. It is located between a DNA fragment. The bacterial moiety is located by attaching to the first insertion DNA fragment and the second insertion DNA fragment, thereby forming a closed circular vector.

The closed loop vector can be used to generate a large amount of plasmid in E. coli, and the plasmid is isolated and cleaved with appropriate restriction enzymes to separate the transforming part from the bacterial part. With the linear transforming portion of yeast DNA, Pichia strains can be transformed for the desired genomic modification.

Of course, the transforming portion of the described closed loop plasmid may also contain additional DNA sequences. For example, bacterial sequences used to produce DNA in vitro using E. coli can be used for transforming DNA, i.e., the bacterial sequence, like the selectable marker gene sequence, can be inserted between the first insert DNA fragment and the second DNA fragment. When using the DNA component of the above composition, the bacterial sequence is also incorporated into the genome of the host yeast used for genomic modification.

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Transformation of Pichia pastoris is described in U.S. Patent No. 4,879,231. Experimental implementation of the transformation of Pichia pastoris will be described later (see Example 1). For the transformation of yeasts of the Pichia strain, a spheroplast is produced by enzymatic cleavage of the cell wall, which is mixed with the transforming DNA and incubated in the presence of calcium ions and polyethylene glycol. It is then regenerated in selective medium. The transforming DNA contains a selective marker gene that can be used to select the cells that have taken up the transforming DNA, since only the transformed cells can live and develop under the particular living conditions used (based on the selective marker used as part of the transforming DNA). . When using a Pichia strain expressing the primary alcohol oxidase gene (AOX1) to express heterologous genes, it is observed that under the control of some promoters (such as AOX1 and / or DAS promoter) the expression of the heterologous gene product is multiplied by using a host containing whole alcohol oxidase. available phrase. Closer examination of the above phenomenon has shown that the expression of heterologous genes in a host can generally be enhanced if a host strain containing a strong substrate-sensitive promoter, wherein this strong substrate-sensitive promoter regulates the heterologous gene, is maintained under substrate conditions under which the promoter is sensitive.

The conditions of the above-mentioned limited viability can be achieved by feeding the cell with a reduced amount of nutrient, or by using a host cell that diminishes viability under certain conditions. For example, if the enhancement of the expression of a heterologous gene is controlled by a methanol-sensitive strong alcohol oxidase or dihydroxyacetone synthase promoter in the medium, conditions providing reduced viability can be achieved and thus the expression of the gene can be enhanced by using a host in which methanol is used. uptake capacity, or using a complete alcohol oxidase gene host but medium methanol medium.

It is believed that the method described above for increasing the expression of heterologous gene products is generally applicable to any organism that has a feeder-reactive promoter, such that placing the heterologous gene under the control of the promoter region and cultivating the host with limited viability promoter, the expression of the gene should increase. Preferably, the limited viability is provided by a mutant host that lacks the ability to degrade nutrients that allow expression of some promoters at a higher level than in a non-mutant host. For the Pichia strain, the above is described in detail in Example 5.

When a strain of Pichia pastoris in which the primary alcohol oxidase gene was disrupted by genomic modification of the invention was cultivated as methanol in carbon, it was surprisingly found that the primary alcohol oxidase deficient strain is still capable of developing in methanol, although to a lesser extent than the original Pichia cells. . This observation indicates that methanol-using Pichia strains have a secondary alcohol oxidase gene.

During the analysis of Pichia chromosomal DNA, a 3 kbp fragment was identified which presumably encodes a portion of the secondary alcohol oxidase gene (AOX2). The fragment was inserted into pBR322 (at the unique BamHI site). The plasmid is designated pPG3.0 and is hosted by E. coli host LE392. This strain was deposited with the Northern Region at the Research Center of the United States Department of Agriculture in Peoria, Illinois under the reference NRRL B-18 022 for public access.

The cleavage map of pPG3.0 is shown in Figure 16b compared to the genomic fragment of the primary alcohol oxidase gauze pPG4.0. The latter fragment is described in detail in U.S. Patent No. 4,885,837. A comparison of the two alcohol oxidase genes (Figure 16) shows that these are not overlapping regions of the same genome. Obviously, there is some homology between the two fragments resulting from the common cleavage sites of the two fragments. Common cleavage sites for the AOX1 and AOX2 genes are indicated by an asterisk in Figure 16. However, both fragments have cleavage sites which do not occur in the other and indicate nucleotide differences between the two fragments.

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

In the examples, the following buffers and solutions were used:

1M Tris buffer 121.1 g of Tris base in 800 ml of water, adjusted to pH with concentrated hydrochloric acid (35% w / w), allowed to cool to room temperature before determination of final pH, final volume Add to 1.

TE buffer 1.0 mM EDTA, 0.01 M pH 7.4 in Tris buffer.

LB (Luria-Bertani) medium 5 g Bacto tryptone, 5 g Bacto yeast extract, 2.5 g NaCl in 1 liter water, adjusted to pH 7.5 with NaOH.

Medium 2B is 0.2 wt% NH₄PO₄, To 1.2 wt₂HPO₄, 0.013% w / w MgSO₄x7 H₂O, 0.074% CaCl₂x2 H₂0.2 µg / ml biotin, 1 µg / ml thiamine, 100 µg / ml tryptophan, 0.4% dextrose, 0.2% casamic acid.

YPD Medium 1% Bacto Yeast Extract 5%

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<td>SD medium</td><td>needle, 2 wt% Bacto peptone, 2 wt% dextrose. 6.75 g of yeast nitrogen base ami-</td>

<td>SED</td><td>without acids (DIFCO), 2% by weight dextrose in 1 liter of water. 1 mol / l sorbitol, 25 mmol / l ED-</td>

<td>SCE buffer</td><td>TA, 50 mM DTT. 9.1 g sorbitol, 1.47 g sodium</td>

<td>CaS</td><td>citrate, 0.168 g EDTA in 50 mL water, pH 5.8 using HCl. 1 mol / l sorbitol, 10 mmol / l</td>

<td>PEG solution</td><td>CaCl₂, sterilized by filtration. 20% by weight polyethylene glycol 3350,</td>

<td>SOS</td><td>10 mM CaCl₂10 mM Tris-HCl (pH 7.4), filtered and sterilized. 1 mol / l sorbitol, 0.3xYPD-</td>

<td></td><td>zeg, 10 mM CaCl₂.</td>

The following abbreviations are used in the examples:

EDTA = ethylenediaminetetraacetic acid

SDS = sodium dodecyl sulfate

DTT = dithio-threitol.

In the examples, enzymatic reactions are performed according to the manufacturer's protocol.

In the case of partial digestion, the time required for proper digestion is determined by digesting at different times, determining the fragments generated by gel electrophoresis, and selecting the time at which the desired size fragment is formed.

First example

Pichia pastoris transformation process

A) Cell culture

First GS115 Pichia pastoris colony (NRRL Y-15,851) was inoculated with about 10 ml YPD medium and the culture was shaken for 12-20 hours at 30 ° C.

Second After about 12 to 20 hours, the cells were diluted to an OD 50 of 0.01 to 0.1 and then maintained in a YPD medium at 30 ° C for about 6-8 hours in a logarithmic development phase.

Third After about 6-8 hours, 100 ml of YPD medium was 0.5 ml OD₆₀₀ = 0.1 (or equivalent) cell culture and shaken at 30 ° C for about 12-20 hours.

4th The culture was harvested by centrifugation at 1500 g / 5 min at an OD 450 = 0.2-0.3 (approximately 16-20 hours).

B) Recovery of spheroplast

First The cells are washed with 10 ml of sterile water (steps 1-5 centrifugation at 1500 g for 5 minutes).

Second The cells were washed with 10 ml of freshly prepared SED.

Third The cells are washed twice with 10 ml of sterile 1 M sorbitol.

4th The cells were resuspended in 10 ml of SED buffer.

5th 5-10 μΐ of 4 mg / ml Zymolyase 60,000 (Miles Laboratories) was added and the cells were incubated at 30 ° C for 30-60 minutes.

Because spheroplast formation is a critical step in transformation, spheroplast formation is controlled by adding 100 μΐ aliquots to 900 μΐ 5% SDS or 900 μΐ 1 M sorbitol solution before or immediately after addition of zymolase, and at different times during the incubation period. The incubation is stopped at the point where the cells are disrupted by SDS but remain intact in sorbitol (usually between 30-60 minutes incubation).

6th The spheroplast is washed twice with 10 ml of sterile 1 M sorbitol at 1000 g / 5-10 min. (The time and speed of centrifugation can be varied, centrifuged until the spheroplast forms a pellet but is not cracked by force.)

7th The cells were washed with 10 ml of sterile CaS solution.

8th All cells were resuspended in 0.6 mL CaS solution.

C) Transformation

First DNA samples (up to 20 μΐ volume) are placed in a 12x75 mm sterile polypropylene tube (DNA is used in water or TE buffer, and approximately 1 μΐ in 5 mg / ml sonicated E. coli DNA is preferred for maximum transformation of small amounts of DNA). t is added to each sample).

Second 100 μΐ of spheroplast was added to each DNA sample and incubated at room temperature for approximately 20 minutes.

Third One ml of PEG solution was added to each sample and incubated at room temperature for about 15 minutes.

4th The samples are centrifuged at 1000 g for 5-10 minutes and the PEG solution is decanted.

5th Samples were resuspended in 150 μΐ SOS and incubated for 30 minutes at room temperature.

6th 850 μΐ sterile 1 M sorbitol was added and an aliquot of the sample was spread as described below.

D) Regeneration of the spheroplast

First Composition of regenerating agar medium:

(a) agar 9 g Bacto agar,

13.4 gKCl,

240 ml H₂O, autoclave;

b) 10X glucose: 20 g dextrose, ml H₂O, autoclave;

c) 10X SC: 6.75 g of yeast nitrogen base without amino acids,

100 ml H₂O autoclave (the desired amino acid or nucleic acid is added up to 200 µg / ml before or after the autoclave);

d) 30 ml of 10X glucose and 30 ml of 10X SC are added to 240 ml of molten agar KC1 solution, 0.6 ml of 0.2 mg / ml biotin and any other desired amino acid or nucleic acid is added up to a concentration of 20 μg / ml, agar was maintained at 55-60 ° C.

Second Spreading Transformed Samples:

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As a bottom agar layer, 10 ml of regeneration agar was added to each plate at least 30 minutes before transformation samples were prepared. 10 ml aliquots of regeneration agar are dispensed into tubes at 45-50 ° C while the transformation sample is in SOS solution. An aliquot of the above transformation samples is added to the regeneration agar in the tubes and poured onto the bottom agar layer on the plates.

Third Determination of the quality of the spheroplast preparation:

pg sample was diluted 100-fold with 990 μΐ 1 M sorbitol. 10 µl of the 100-fold dilution was diluted 100-fold again with a second portion of 990 μΐ 1 M sorbitol. 100 μΐ of each dilution was plated on YPD-containing agar plates to determine the concentration of whole cells remaining in the preparation without spheroplast. 100 μΐ of each dilution was added to 10 ml regeneration agar containing 40 pg / ml histidine to determine total recoverable spheroplast. The value to be used for transformation is 1-3x10⁷ total recoverable spheroplast / ml and about 1x10³ total cells / mL.

4th Plates were incubated at 30 ° C for 35 days.

Second example

Site-specific insertion of Saccharomyces ARG4 gene and pBR322 and deletion of Pichia HIS4 in GS190 (NRRL Y-18014)

Figure 1 depicts plasmid pYMI1 and Figure 2 illustrates the site-specific insertion process of the plasmid into the P. pastoris gene. The vector inserts the Saccharomyces ARG4 gene and the pBR322 DNA sequence into the Pichia HIS4 site, simultaneously deleting the entire HIS4 gene site from the Pichia genome. Other sequences, such as expression sequences, can be readily incorporated into pYMI1 and with it into the P. pastoris genome.

To construct plasmid pYMI1, plasmid pYJ8 from E. coli deposited under accession number NRRL B15889 (Figure 3) was partially digested with EcoRI and Clal to give a 400 bp EcoRI / Clal fragment. In parallel, the same plasmid was completely cleaved with BamHI and partially cleaved with EcoRI to give an approximately 400 bp EcoRI / BamHI fragment.

These two 400 bp fragments and Eco RI were ligated via a sticky end to an 800 bp fragment containing the 5 'and 3' ends of the Pichia HIS4 gene.

The commercially available plasmid pYM25 (Figure 17) from E. coli (NRRL B-18015) was partially digested with HindIII and SalI. The resulting 2.6 kb fragment containing the ARG4 gene of Saccharomyces cerevisiae was isolated. The ARG4 gene encodes the enzyme arginine succinate lyase. The isolated 2.6 kb fragment was used as a selectable marker.

The 800 bp fragment and the 2.6 kb fragment were ligated into pBR322 via the ClaI / BamHI site and the HindIII / SalI site.

The arginine prototrophy was transformed with P. pastoris arg4 strain GS190 (NRRL Y18014) with EcoRI-cleaved pYMI1 (Arg⁺). 40 µg / ml histidine is added to the regeneration agar medium to select for transformants that require histidine for grinding the HIS4 gene.

The Arg⁺ of the transformants we select His-like due to deletion of the HIS4 gene. For selection of His transformants, the embedded Arg⁺ colony regeneration agar is transferred to a 50 ml sterile tube containing 25 ml sterile water. The agar is pulverized with a Brinkman Instruments Polytron homogenizer at speed 5 for about 1 minute. The yeast cells were separated from the agar by filtration through three layers of gauze. The yeast cells are then sonicated, diluted and plated on SD medium agar plate containing 40 pg / ml histidine.

For ultrasound treatment, the cell sample is diluted to A ^ q = 0.1 and treated for 4 seconds with Sonifier Cell Disrupter 350 (Branson Sonic Power Co.), which promotes cell separation but does not reduce cell viability. . After 2-3 days, colonies developing on SD + histidine agar plates are plated on histidine and non-histidine SD plates.

The Arg⁺ The proportion of HIS 'colonies averaged 0.7% of the total Arg⁺ relative to the transformant. 3 Arg⁺ HIS 'strain genomic DNA was analyzed by Southern blot hybridization. All three samples lack the complete HIS4 gene and incorporate the linear plasmid as shown at the bottom of Figure 2.

In addition to requiring histidine and no need for arginine for the development of the GS190-pYMI1 strain, nutritional requirements and other developmental parameters are changing.

Third example

Deletion of the HIS4 gene from P. pastoris NRRL Y-1130

Figure 4 shows the process of linear insertion of plasmid pYMI3a; Figure 5 shows the process of linear insertion of the plasmid fragment into the HIS4 site of P. pastoris NRRL Y-1130.

To construct plasmid pYMI3a, plasmid pBPG1-1 in strain E. coli deposited with NRRL B18020 (Figure 6) was partially digested with BamHI and BglII and the 2.2 kb fragment containing the G418 resistance gene was isolated.

Plasmid pYJ8 was cleaved with BglII and the central 2.7 kb portion of the HIS4 gene was removed. The resulting 2.2 kb fragment was ligated into the BglII site of the resulting fragment.

The pYMI3a vector was digested with EcoRI to give a 2.9 kbp fragment containing the G418^R gene to which approximately 450 and 250 bp DNA is attached at both ends, resulting from the 5 'and 3' ends of the HIS4 gene. The EcoRI-cleaved plasmid was transformed into a P. pastoris host. Transformants are selected for their ability to grow in the presence of 300 pg / ml antibiotic, this G418^r transformed. G418^R to select the re7

Generation B agar medium is modified as described in Example 1 (d) as follows:

First Composition of regeneration agar medium:

(a) agar-sorbitol 9 g Bacto-agar,

54.6 g of sorbitol,

240 ml H₂O autoclave;

b) 10x glucose, 20 g dextrose

100 ml H₂O autoclave;

c) 10xYP ', 10 g yeast extract, g peptone,

100mlH₂0, autoclave;

d) 30 ml of 10x glucose and 30 ml of 10x YP are added to 240 ml of molten agar sorbitol solution, the regeneration agar medium is maintained at 55-60 ° C.

Second Distribution of transformants:

At least 30 minutes before the transformation samples are prepared, 10 ml of regeneration agar medium containing 600 pg / ml G418 is plated on the bottom of the plate. While the transformation samples are in SOS medium, 10 ml aliquots of regeneration agar medium (without G418) are added to tubes at 40-50 ° C bath temperature. After the transformation samples were prepared, an aliquot of the samples was added to the regeneration agar in the tubes and poured onto a 10 ml bottom agar layer containing G418. The plates were incubated at 30 ° C for 3-5 days.

After colonies develop on regeneration agar plates containing G418, cells are assayed for their ability to grow without histidine. Cells were extracted from regeneration agar, sonicated and plated on SD medium agar plate containing 40 pg / ml histidine as described in Example 2. After incubation at 30 ° C for 2 to 3 days, colonies are plated onto histidine-containing SD medium without histidine.

G418^R about 0.1% of colonies (2 of about 2,000 tested samples) are His. Both His strains lack the HIS4 gene and both genomes contain G418 by Southem blot hybridization.^R gene as shown in Figure 5. One of the His' strains that receives the KM31 laboratory signal (United States Department of Agriculture, Northern Region Research Center, Peoria, Illinois, NRRL Y-18018) can be successfully transformed with a number of HIS4-containing Pichia-based plasmids, such as pSAOH5 (produced by NRRL B). -15,862 from E. coli), which again indicates that KM31 is an organism capable of deleting the HIS4 gene.

This is the first case that the "wild-type" P. pastoris NRRL Y-11 430 can be directly transformed (i.e., without first isolating and characterizing an auxotrophic derivative). The potential advantage of HIS4 mutant strains, despite being produced by site-specific insertion / deletion, is that they are free of secondary mutations that occur in, for example, chemical mutations in Pichia auxotrophic strains, such as GS115 (NRRL Y-15851) and GS190 ( NRRL Y-18 014).

Note that replacement of the Pichia HIS4 gene from the Pichia genome with an EcoRI fragment from pYMI3a does not result in the incorporation of a large amount of pBR322 (as occurs in pYMI1, Example 2). Because most ARS-based Pichia expression vectors, such as pSAOH5 (Figure 9), are primarily derived from the pBR322 sequence and the Pichia HIS4 gene, these autonomic vectors show little homology to hosts without Pichia HIS4 and therefore do not integrate frequently.

4th example

Destruction of the primary alcohol oxidase gene

Pichia strains without the alcohol oxidase gene (the primary alcohol oxidase gene is designated AOX1 and the secondary alcohol oxidase gene is designated AOX2) are significant for at least two reasons. First, they promote the study of the regulation of gene expression by methanol. For example, the mutant strain AOX1 and AOX2, which is described in detail in Example 7, can be used to determine whether methanol or its other metabolites (formaldehyde, formic acid, etc.) induce methanol-regulated genes. The second significance of the Pichia strain without AOX is to allow high levels of expression of the heterologous gene products described in Examples 5 and 6.

Plasmid pYMI7 ​​(Figure 7) was constructed to disrupt the AOX gene. For this, plasmid pYM25 (NRRL B-18015, Figure 17) was partially digested with SalI and completely digested with BamHI. The resulting 2.9 kb BamHI / SalI fragment contains the ARG4 gene of Saccharomyces cerevisiae.

Plasmid pPG4.0 (NRRL B-15868, Figure 16a, cleavage map of the Pichia portion of the plasmid) was digested with BamHI and ligated with the above 2.9 kb fragment. Because the SalI end of the 2.9 kb fragment does not correspond to the BamHI end, Klenow is used to "load" after ligation of the BamHI ends, then the blunt end of the former SalI site of the fragment and the former BamHI site of the vector. ligated.

The resulting plasmid pYMI7 ​​results in the insertion of approximately 600 bp (about 1/4 of the gene) of the 5 'region of the AOX1 gene. Plasmid pYMI7 ​​was digested with PvuII and EcoRI under standard conditions and⁺ prototrophy by transformation into PPF1 (arg4, his4, NRRL Y-18017). The transformants were extracted from regeneration agar and sonicated as described in Example 2, and finally plated on SD medium agar plates containing 0.1% glucose (instead of 2%) and 40 pg / ml histidine. The resulting colonies are transferred to SD medium agar plates (containing histidine) containing the following carbon sources:

First without coal,

Second 0.5% methanol and

3.2% glucose.

The Arg⁺ about 81.0% of colonies are unable to develop normally in methanol. Does not use methanol

In two of the colonies HU 208,552 Β, KM71 and KM72, Southern blot analysis was performed on genomic DNA to determine that the AOX1 gene was disrupted and that the vector was inserted, as can be seen at the bottom of Figure 8.

The genotype of PPF1-pYMI7 ​​alcohol without oxidase is (his4 aoxl:: SARG4), which is very valuable. For example, since the strain is his4, a Pichia vector containing the HIS 4 gene as a selectable marker, such as pSAOH5 (NRRL B-15862, Figure 9), it can be transformed into the host organism as described in Example 5.

5th example

Methanol-regulated expression of the lacZ gene in Pichia pastoris mutant strain without alcohol This example is an example of the lacZ gene produced in Example 4.

KM71 describes the expression of Pichia host (PPFI-pYMI7 ​​alcohol without oxidase).

For comparative studies, KM71 (his4 aoxl:: SARG4) and Aoxl as host Aoxl⁺ as host, PPF1 (arg4, his4; NRRL Y-18017). The AOX1 promoter-lacZ expression region was transformed into both strains on plasmid pSAOH5 (Figure 9, NRRL B15862). Several stable His + transformants were isolated from both strains. Their genomic DNAs were analyzed by Southern blot analysis to obtain a set of strains containing pSAOH5 linked at the A0X1 promoter site. Like the living, the dihydroxyacetone synthase (DAS) promoter-lacZ gene fusion was transformed into KM71 and PPF1 by plasmid pT76H3 (Figure 18, NRRL B-18000) by histidine prototropic selection.

All four strains develop on SD medium, except that it contains 2% glycerol as the sole carbon and energy source instead of 2% glucose. The cultures were centrifuged and then transferred to SD medium containing 0.5% methanol as the sole carbon source. Samples were assayed for β-galactosidase and the results are shown in Table 1.

First β-galactosidase, units / gg *

<td>Time (h)</td><td>aoxl ⁺</td><td>farmer</td><td>aoxl</td><td>farmer</td>

<td>promoter</td><td>AOX1</td><td>DAS</td><td>AOX1</td><td>DAS</td>

<td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 2</td><td> 3</td><td> 2</td><td> 5</td><td> 4</td>

<td> 4</td><td> 11</td><td> 8</td><td> 7</td><td> 7</td>

<td> 10</td><td> 18</td><td> 9</td><td> 12</td><td> 11</td>

<td> 16</td><td> 17</td><td> -</td><td> 26</td><td> -</td>

<td> 20</td><td> 21</td><td> 12</td><td> 38</td><td> 18</td>

<td> 25</td><td> -</td><td> 16</td><td> -</td><td> 24</td>

<td> 30</td><td> 18</td><td> -</td><td> 43</td><td> 27</td>

<td> 32</td><td> -</td><td> 22</td><td> -</td><td> 37</td>

<td> 42</td><td> 14</td><td> -</td><td> 72</td><td> -</td>

<td> 50</td><td> -</td><td> 22</td><td> -</td><td> 48</td>

<td> 54</td><td> 14</td><td> -</td><td> 48</td><td> -</td>

- β-galactosidase assay

A) Solvents used

<td>Z-buffer:</td><td colspan="2">Final concentration</td>

<td>So₂HPO₄x7 H₂SHE</td><td>16.1 g</td><td>0.06 mol / l</td>

<td>NaH₂PO₄</td><td>5.5 g</td><td>0.04 mol / l</td>

<td>KC1</td><td>0.75 g</td><td>0.01 mol / l</td>

<td>MgSO₄x7 H₂SHE</td><td>0.246 g</td><td>0.001M</td>

<td>2-mercaptoethanol</td><td>2.7 ml</td><td>0.05 mol / l.</td>

Make up to 1 liter and adjust the pH to 7.

Second O-Nitrophenyl 4-D-galactoside (ONPG)

400 mg of ONPG (Sigma N-1127) in 100 ml of distilled water gives 4 mg / ml of ONPG.

B. Test procedure

First An aliquot of the culture medium (20-50 OD₆₀₀yeast cells) and the cells are washed with cold sterile water.

Second Add 1 μΐ of 40% Z-buffer and 0.2 g of acid-washed 0.45-0.50 mm glass beads. The samples were placed on ice and stirred vigorously twice for 1 minute. Between the mixing phases, the samples are kept on ice for at least 1 minute.

Third The extracts were placed in microcentrifuge tubes and centrifuged at 4 ° C for 5 minutes. The supernatant of freshly centrifuged samples was separated and the extract was kept on ice.

4th The total protein concentration of the extract was determined by the BioRad Laboratories (Bradford) protein assay. To do this, the Bio-Rad dye reagent concentrate was diluted with 4 volumes of deionized water and filtered on Whatman 3MM paper. To record the standard concentration curve, 3, 10, 30 and 100 g of bovine serum albumin (BSA) in 100 µl of Z buffer are added to 13 100 mm glass tubes containing 2.5 ml of dye reagent. The samples were mixed and kept at room temperature for 5 minutes, and the optical density was measured at 595 nm. 3, 10 and 30 µl of the extract were analyzed in 100 µl of Z-buffer as described above. The protein concentration of the extracts was determined by interpolation on the BSA concentration curve.

5th For the β-galactosidase assay, 10 µl of a ten-fold diluted extract was added to 1 mL of Z buffer and incubated for 5 minutes at 30 ° C.

6th The reaction was started by adding 0.2 mL of ONPG (4 mg / mL) and stirred.

7th The reaction was treated with 0.5 mL of 1M NaOH₂CO₃ solution at the desired time (usually 1-30 minutes)_42o&lt;1) stopped.

8th The absorbance of the supernatant was examined at 420 nm.

C) Determination of β-galactosidase activity unit

One unit (U) corresponds to the formation of 1 nmole of orthonitrophenol (ONP) per minute at 30 ° C and pH 7.

nmol ONP absorbance at 420 nm (A₄₂₀The absorbance 1 at 0.0045 at 1 cm, i.e. 420 nm, corresponds to 222 nm ONP / ml or 378 nm / 1.7 ml since the supernatant analyzed volume is 1.7 ml. Accordingly, the unit is calculated using the following formula:

HU 208 552 Β

U = ^A42- ~ χ 378 h (min)

All 4 samples show barely detectable β-galactosidase activity during the glycerol developmental phase. About 10-20 hours after transferring to methanol, Aoxl⁺ two cultures containing AOX1-lacZ and DAS-lacZ expressing host have β-galactosidase activity at about 20 units / pg protein. The activity of the sample containing AOX1-lacZ in Aox1 is 60 units / pg. In the Aoxl host, the β-galactosidase activity of the sample containing the DAS-lacZ moiety was also increased, i.e., it expresses the transformed isoxic Aoxl at 2-3 fold levels of the transfected Aoxl host, KM71.⁺ strain, relative to PPF1.

6th example

Insertion of the Hepatitis B surface antigen gene and

Deletion of the AOX1 gene

In this example of site-specific insertion / deletion, the entire coding sequence of the AOX1 gene is deleted and the Hepatitis B surface antigen (HBsAg) gene is inserted using the AOX1 gene promoter, which remains in the genome. Plasmid pBSAGI5I (deposited in E. coli) was prepared for this P. pastoris host. coli host in the Northern Region at the Research Center of the United States Department of Agriculture, Peoria, Illinois, and unrestricted to the public after the patent application of this application has been granted, No. NRRL B-18 021, Figure 12). The plasmid contains a 1.0 kbp fragment and a sequence linked to the 5 'end of the AOX1 gene, followed by a Hepatitis B surface antigen (HBsAg) sequence and a 300 bp AOXI1 terminator fragment (Figures 9, 10 and 11). The expression region is followed by a 2.7 kbp fragment encoding the Pichia HIS4 gene and a 1.5 kbp PvuII fragment containing the 3 'sequence of the AOX1 gene. Digestion of pBSAGI5I with BglII yields a 7.2 kbp linear vector containing 0.85 kbp 5'-AOX1 gene sequence at one end and 1.1 kbp 3'-AOX1 sequence at the other end. BglII-cleaved pBSAGI5I is transformed into GS115 by selection of histidine prototrophy, the transformants are extracted from regeneration agar and sonicated as in Example 2 and finally containing 0.1% glucose (instead of 2.0%). Plate onto SD medium. The resulting colonies are transferred to minimal agar plates containing as carbon source:

First without coal,

Second 0.5% methanol,

Third 2% by weight glucose.

On average, 32% of colonies tested were unable to develop normally on methanol.

Two of the non-methanol colonies performed Southem spot analysis on genomic DNA showing that the AOX1 gene was deleted and the vector sequence was inserted (Figure 13).

When grown in methanol, the GS115-pBSAGI5I strain (AOX1: HBsAg-HIS4) expresses HBsAg at a higher level than cells with similarly transformed whole alcohol oxidase.

7th example

Identification of the secondary alcohol oxidase gene of P. pastoris by site-specific modification

The presence of the secondary alcohol oxidase gene can be inferred from the following observations:

First Southern blots in which the AOX cDNA or genomic DNA samples were hybridized with limited Pichia genomic DNA always show at least 2 bands.

Second Two Pichia genomic DNA fragments were isolated that are similar but not identical (Figure 16).

Third Mutant Pichia strains, such as KM71 and GS115pBSAGI5I, in which the primary AOX gene (AOX1) is deleted or destroyed, develop methanolone and exhibit alcohol oxidase activity.

The development rate of AOX strains developing methanol in cells is much lower than that of the isogenic AoxX⁺ strains. Thus, the secondary AOX gene (AOX2) appears to be developed at a much lower level or its products are less active in methanol. THE

4th Example 1A shows that the Pichia DNA fragment contains the AOX1 gene in pPG4.0.

The most convincing method of demonstrating that the DNA fragment of the pPG3.0 genome contains at least a portion of the AOX2 gene is to produce a mutant strain in which the original AOX2 gene is disrupted or deleted. For this, a site-specific vector pXMI12a (Figure 14) was constructed. The plasmid is primarily composed of pPG3.0 (Figure 16b), which contains the original AOX2 gene on a 3.0 kbp BamHI fragment. The 2.7 kbp BglII fragment containing the Pichia HIS4 gene from pYJ8 (Figure 3). Figure 7, NRRL B-15889) and inserted at the site of the sequence located between the BglII and left KpnI sites of pPG3.0. (The KpnI site is converted to the BglII site using an oligonucleotide adapter before insertion.

The adapter required for shaping can be easily selected by one of ordinary skill in the art, knowing the old (KpnI) and new (BglII) cleavage sites. The method may be implemented, for example, as shown in FIG.

The BamHI site of the HIS4 gene is disrupted by "filling in" before being inserted into pPG3.0 according to Figure 20. A comparison of the AOX1 and the original AOX2 genes (Figure 16) shows that this construct was deleted by approximately 800 bp. is obtained in the middle of AOX2. Digestion of pXMI12a with BamHI results in a 4.5 kbp linear vector containing the HIS4 gene fragment to which the 1.1 and 0.7 kbp sequences from the original AOX2 site are attached (Figure 15).

This linear vector was transformed into AOX1 strain KM71 (AOX1 HSI4: SARG4) and transformants were isolated by histidine prototrophy selection. Transformants are assayed for methanol utilization by transplanting onto agar plates.

The KM71 untransformed Aoxl strain grows so slowly on methanol plates that

HU 208 552 Β volatiles evaporate before significant growth can be observed. To solve this problem, the cells are fed with methanol in the vapor phase. To do this, about 0.2 ml of 100% methanol is placed under the plates which do not contain a carbon source in the agar medium. The plates were allowed to stand at room temperature and fresh methanol was replaced in the lower portion every 24 days. After about 1-2 weeks, the difference between the wild type (Aoxl⁺, Aox2⁺) and mutant strains [(Aoxl^- Aox2⁺) and AoxlAox2 "] methanol.

After steam phase feeding, we find that the Aoxl strains are His⁺ about 0.1% of its transformant is unable to grow on methanol. 8 Aoxl^-Aox2^- His^- transformant DNA was analyzed by Southem hybridization. 3 DNA contains the inserted linear pXMI12a vector (Figure 15). An Aoxl^-Aox2^- analysis of the double mutant KM7121 (NRRL Y-18,019) shows that the strain does not develop at all in methanol and has no detectable AOX activity. Based on the above property of KM7121, it is obvious that Pichia ffagmens contains the sequence of the secondary AOX gene in pPG3.0 and that, apart from the two alcohol oxidases above, P. pastoris does not show any other methanol oxidizing activity.

The foregoing examples merely illustrate the applicability of the invention and are not to be construed as limiting the scope thereof. Possible variations and modifications that do not affect the spirit of the invention are within the scope of the invention.