KR100267666B1 - Plasmid vectors for pichia ciferrii - Google Patents

Abstract

PURPOSE: Provided is an expression plasmid for Pichia ciferrii to insert targeted genes as many as possible into the strain when transformation of Pichia ciferrii is required. CONSTITUTION: The expression plasmid for Pichia ciferrii is obtained by the next steps of: i) separating the gene of ribosome protein L41 of sequence ID. No. 1 from Pichia ciferrii; ii) making the L41 gene resistant to cycloheximide and manufacturing plasmid pCYH1.9¬r to select transformants; iii) separating ribosome DNA and making plasmid prDX9.0 to insert targeted genes into chromosome more effectively; and iv) making a recombinant plasmid by treating ribosome DNA with restriction enzyme.

Description

Expression Plasmids for Pchia Ciferi

The present invention relates to an expression plasmid for Pichia ciferrii, and more particularly, to a ribosome DNA derived from Pichia ciferi, a CYH ^r gene having resistance to antibiotic cycloheximide, and an object It relates to a plasmid consisting of genes.

Pizzia ciferi is a biological desulfurization of coal (Stevens et al., US Pat. No. 4,851,350), production of D-α-amino acids (Takeichi et al., US Pat. No. 5,068,187), (S) -1-phenyl-1,3- It can be used for the production of propanediol (Ajinomoto, Japanese Patent Laid-Open No. 6-90789), the production of secondary alcohols by stereospecific ketone reduction (Merck, EP-300287), and especially the raw material of ceramide Tetraacetyl phytosphingosine (TAPS: tetraacetyl phytosphingosine) is produced and secreted out of the cell and has high utility (Barenholz et al., Biochem. Biophys. Acta, 248, 458, 1971; ibid, 306, 341 , 1973).

Phytosphingosine including TAPS, like its own ceramide, not only shows effective effects such as protecting skin and protecting skin from drying, but is also produced by various microorganisms, making it easy to secure natural raw materials. It is also useful as a raw material of ceramide because it is easily converted into ceramide. For this reason, phytosphingosine or ceramide, including TAPS, have all been used in functional cosmetics for the purpose of protecting the skin.

However, the TAPS production of wild strains of Peachia ciperi ATCC 14091 and F-60-10 (NRRL 1301) was very low (Wickerham & Burton, J. Bacteriol., 80, 484, 1960). Mutant strain development to increase is actively underway (Wickerham & Burton, J. Bacteriol., 80, 484, 1960; US Pat. No. 5,618,706). The present inventors have also filed a patent application for developing a mutant strain (KFCC-10937) with a significant increase in productivity due to increased TAPS production and a shorter fermentation period (Korean Patent Application 96-67997).

Furthermore, the present inventors have approached genetic engineering to maximize the usability of the chia ciferi. However, pichia ciferi was called the genus Hansenula, and was recently renamed the genus pichia by 5S-RNA analysis (Yamada et al., Biosci. Biotechnol. Biochem., 58, 1245, 1994), genetic studies on the Peach subfamily are insufficient. Therefore, no transformation method has been developed for the Pchia ciferi strain.

In this situation, the present inventors used a transformation method for Candida utilis (Kondo et al., J. Bacteriol., 177, 7171, 1995) to perform transformation for pichia ciferi. Developed. As a result, the DNA sequence was determined by cloning the ribosome protein L41 gene of Peachia ciperi, and the marker gene for selection of transformants was given to this gene by giving resistance to the antibiotic cycloheximide derived from yeast. When the target gene is transformed into pichia ciferi using the plasmid inserted into the ribosome gene of pichia ciferi as an expression vector, the insertion efficiency of the target gene into the chromosome can be maximized. This invention was completed.

Accordingly, it is an object of the present invention to provide genetic information for the ribosome protein L41 gene of pichia ciferi.

Another object of the present invention is to provide an expression plasmid for pichia ciferi consisting of ribosomal DNA derived from pichia ciferi, L41 gene resistant to antibiotic cycloheximide, and a target gene.

In addition, another object of the present invention is to provide a method for transforming Pchia ciferi into a gene of interest using the above-described expression plasmid.

1 is a restriction map of a plasmid in which the L41 gene derived from peach ciferi is inserted into various sites of the ribosomal gene, respectively,

The hatched portion is ribosomal DNA,

Restriction enzyme shown in bold letters is the restriction enzyme used to open each plasmid,

The transcription direction of each gene is indicated by an arrow,

In the figure, the plasmid prACL2 contains the LCB2 gene encoding serine palmitoyl transferase derived from pichia ciferi as a gene of interest.

Transformation method for the chia ciferi according to the present invention was developed using the transformation method for Candida utilis (Kondo et al., J. Bacteriol., 177, 7171, 1995) will be. In this method, in consideration of expression in yeast, instead of antibiotic resistance marker genes such as kanamycin derived from bacteria, antibiotic resistance marker genes derived from yeast are used.

To this end, first, the nucleotide sequence of cloned ribosome protein L41 of chia ciferi is determined to determine the base sequence, and from this base sequence, amino acid No. 56 showing sensitivity to the cycloheximide, an antibiotic derived from yeast, is proline. Confirmed. By replacing proline, amino acid 56, with glutamine, L41 protein was given resistance to cycloheximide and used as a marker gene for selection of transformants.

1. Isolation of the Ribosomal Protein L41 Gene of Pchia Ciferi:

Primers CYH1 and CYH4 synthesized with reference to Condo et al. (Kondo et al., J. Bacteriol., 177, 7171, 1995):

CYH1: 5'-CGC GTA GTT AAY GTN CCN AAR AC-3 '

CYH4: 5'-GCC TGG CCY TTY TGY TTY TTN TC-3 '

Using a PCR method, a fragment of about 300 bp in size containing the L41 gene was isolated from the genomic DNA of the chia ciferi, and the probe was prepared by labeling the fragment.

Using this probe, Southern blot with genomic DNA of Peachia ciperi was used to identify the band at 1.9 kb position of genomic DNA treated with restriction enzyme EcoR I, and the 1.9 kb EcoR I DNA fragment was extracted to obtain plasmid pCYH1.9. After preparation, the DNA sequence was determined, and the results are shown in SEQ ID NO: 1. DNA sequence of the L41 gene of Peachia ciperi ATCC 14091 was registered as AF 053457 (98.3.7) in GeneBank.

The L41 gene of Pchia ciferi ATCC 14091 consists of 737bp including 419bp intron, and the amino acid sequence is estimated based on the nucleotide sequence, and shows 90% or more similarity with other yeasts. The 56th amino acid, which is sensitive to the antibiotic cycloheximide, was identified as proline.

2. Imparting resistance to cycloheximide to L41 gene: Construction of plasmid pCYH1.9 ^r for transformant selection:

Site directed mutagenesis was used to replace proline No. 56 with glutamine to confer resistance to the antibiotic cycloheximide. A plasmid pCYH1.9 ^r was constructed.

Hereinafter, in the present specification, the L41 gene may be referred to as CYH, and the L41 gene showing resistance to cycloheximide may be referred to as CYH ^r .

3. Isolation of ribosomal DNA: Construction of plasmid prDX9.0 to increase the efficiency of insertion of the target gene into the chromosome:

In order to increase the efficiency of gene insertion into the chromosome, ribosome DNA having hundreds of copies in the cell is used.

PCR reaction with genomic DNA of Peacha ciperi ATCC 14091 using primers synthesized with reference to partial sequencing of Peacha ciperi ribosomal RNA (Yamada et al., Biosci. Biotechnol. Biochem., 58, 1245, 1994) The ribosomal DNA fragment of 6.0 kb size was isolated, and this fragment was Southern blotted with a probe to separate the ribosomal DNA fragment of 9 kb Pchia ciferi ATCC 14091, and inserted into the plasmid pBluescript KS + to prepare plasmid prDX9.0. .

Partial nucleotide sequences of 9 kb ribosomal DNA fragments were determined to confirm the position and orientation of 5S, 26S, 5.8S, and 18S ribosomal protein genes as shown in FIG. 1.

4. Construction of Recombinant Plasmids:

Ribosome DNA is treated with various restriction enzymes,

1) Review of correspondence with transcription direction

2) Arrangement method of ribosomal DNA and CYH ^r gene

3) Examination of transformation efficiency when using ribosomal RNA structural gene or non-transcribed region of ribosomal DNA as introduction site

Various plasmids were prepared as shown in FIG. 1 by connecting with CYH ^r so as to reflect the concept.

The properties of the plasmid of Figure 1 are summarized in Table 1.

Plasmid Characteristics prXHNC The 3.5 kb ribosomal DNA obtained by treatment with Xba I was further treated with Hpa I / Nco I to remove 1.6 kb of ribosomal DNA, and CYH ^r was inserted. prEHC 3.8 kb of ribosomal DNA obtained by treatment with EcoR I was further treated with Hpa I to remove 2 kb of ribosomal DNA and CYH ^r was inserted. prCEX Binding CYH ^r to the EcoRⅠ position of 1.1kb ribosomal DNA obtained by treatment with EcoRⅠ / XbaⅠ. prCRX Binding CYH ^r to the EcoRV position of 1.3kb ribosomal DNA obtained by treatment with EcoRV / XbaI. prXCH Insert CYH ^r into Hpa I position of 3.5kb ribosomal DNA obtained by treatment with Xba I. prXCE Insert CYH ^r into the EcoRⅠ position of 3.5kb ribosomal DNA obtained by treatment with XbaI. prXHC1.9 Insert CYH ^r into the EcoRⅠ position of 1.6kb ribosomal DNA obtained by treatment with XhoⅠ / HindIII. prAC1.9 Binding CYH ^r to the EcoRV position of 0.6kb ribosomal DNA obtained by treatment with HindIII / EcoRV. prEC1.9 Binding CYH ^r to the EcoRⅠ position of 1.4kb ribosomal DNA obtained by treatment with HindIII / EcoRⅠ. prHEC1.9F Insert CYH ^r into the EcoRV position of 1.4kb ribosomal DNA obtained by treatment with HindIII / EcoRI. prHEC1.9R Reverse direction of CYH ^r in prHEC1.9F

Introducing nomenclature, for example, in plasmid prAC, p means plasmid, r means ribosomal DNA, and C means CYH ^r . A and E of prAC1.9 and prEC1.9 refer to restriction enzymes that cleave ribosomal DNA to open the plasmid. For other plasmids, X is XbaI, E is EcoRI, R is EcoRV, H means that the ribosome DNA is treated with HindIII restriction enzyme, and the position of C (CYH ^r ) is the sequence of alignment between ribosomal DNA and CYH ^r Means.

5. Transformation and selection of transformants:

According to the class and Peter's method (Klass & Peter, Curr. Genet., 25, 305, 1994), plasmids were added to the parent strain recovered by centrifugation when the absorbance at 600 nm was raised to 1.5, followed by a voltage of 500 mA and an electrostatic After electroporation was applied under conditions of a capacity of 50 kPa and a resistance of 800 kPa, a transformant was selected by spreading onto a YEPD solid medium containing cycloheximide.

As a result of comparing the transformation efficiency (Table 2 in Example 17), the plasmid prHEC1.9F using the non-transcribed site between the 5S and 26S ribosomal RNA structural genes was the most efficient, and 5S RNA as in the plasmid prHEC1.9R. When the transcription direction of the gene and the transcription direction of the CYH ^r gene were reversed, the transformation efficiency decreased significantly.

6. Transformant Analysis by Southern Blot:

Genomic DNA was extracted from the transformants and the introduction pattern of the L41 gene was analyzed by Southern blot method, and it was confirmed that 4 to 5 copies of the L41 gene were present in the genomic DNA of the transformant. From this, it can be seen that when the target gene using the expression plasmid provided in the present invention is transformed into Pchia ciferi, the insertion efficiency of the target gene into the chromosome can be maximized.

7. Production of TAPS by Preparation, Transformation and Fermentation of Transformant Plasmid prACL2 for TAPS Production:

Utilizing the transformation method developed above, the LCB2 gene encoding serine palmitoyl transferase, an enzyme involved in the biosynthesis of TAPS, as a target gene was transferred to the mutant strain pichia ciferi KFCC-10937 developed by the present inventors. Transformed.

7-1. Isolation of the gene LCB2, which encodes serine palmitoyl transferase:

Using primers synthesized by referring to the DNA sequence of the LCB2 gene of Saccharomyces cerevisiae (Nagiec et al., Proc. Natl. Acad. Sci. USA, 91, 7899, 1994). LCB2 gene was isolated from Saccharomyces cerevisiae, and a 0.9kb SalI fragment of the isolated LCB2 gene was used as a probe to Southern blot from genomic DNA of Peachia ciperi to contain a 12kb LCB2 gene. The DNA band was confirmed, and Southern blotting was repeated to reduce the size of the 3.0 kb ScaI / AflIII fragment to prepare plasmid pL2SA. The base sequence of the LCB2 gene was determined, and the result is shown in SEQ ID NO: 2. DNA sequence was registered in GenBank as AF053456 (98.3.7).

Comparing the nucleotide sequences, the LCB2 gene of the Pchia ciferi ATCC 14091 was 1688 bp in size, without introns, and the amino acid sequence inferred based on the nucleotide sequence was similar to that of Saccharomyces cerevisiae. In addition, a transmembrane helix region is present at positions 55-79, and the same amino acid sequence is present in a region including pyridoxal phosphate and lysine forming a chiffon base. Had.

7-2. Construction of the plasmid prACL2:

A plasmid prACL2 was prepared by inserting a 3.0kb LCB2 fragment obtained by treating plasmid pL2SA in the order of restriction enzymes HindIII / Klenow / BamHI into plasmid prHEC1.9F treated with restriction enzyme Eco47III / BamHI.

The plasmid prACL2 prepared above is in the form of ribosomal DNA, CYH ^r (L41), and LCB2 genes.

The plasmid prACL2 obtained above was internationally deposited on 4 May 1998 under the provisions of the Budapest Treaty in the Gene Bank of the Institute of Biotechnology, Korea Advanced Institute of Science and Technology, and was assigned accession number KCTC-0468BP.

7-3. Transformation:

This plasmid was transformed into the mutant strain Peach ciferi KFCC-10937 developed by the present inventors according to the method described in 5. above to select a transformant having a high gene copy number.

7-4. Production of TAPS by Fermentation of Transformants:

The transformant was optimized for YGM medium (glycerol 100g / l, yeast extract 2g / l, KNO ₃ 3g / l, (NH ₄ ) ₂ SO ₄ 0.5g / l, MgSO ₄ 7H ₂ O 0.3g / l, NaCl 0.5 g / l, CSL 3g / l, LS-300 1g / l) to ferment for 4 days to produce TAPS.

The transformant provided by the present invention has at least 1.3 times the TAPS production capacity as compared to the parent strain KFCC-10937.

Hereinafter, with reference to the embodiment will be described in more detail the configuration and effect of the present invention.

<Example 1> Extraction of genomic DNA of Pchia ciferi ATCC 14091:

Peach Ciferri ATCC 14091 recovered by incubation in YEPD medium (2% peptone, 1% yeast extract, 2% glucose) according to Johnston's method (Yeast Genetics, molecular aspects, pp. 107-123, IRL Press, 1988). The suspension was suspended in an SSEM solution (1M sorbitol, 100 mM sodium citrate, 60 mM EDTA, 100 mM 2-mercaptoethanol). Protoplasts were prepared by adding 0.1 mg of Novozyme per ml of the suspension, followed by treatment at 37 ° C. for 30 minutes. An equal volume of SDS-TE solution (2% SDS, 50 mM EDTA in 1M Tris-Cl, pH8.0) was added, followed by reaction at 60 ° C. for 10 minutes to disrupt the cells. The supernatant obtained by centrifugation at 8,000 rpm for 15 minutes was precipitated by adding twice the volume of ethanol, dissolved in TE solution, and then treated with RNase to extract genomic DNA of pichia ciferi.

<Example 2> Isolation of L41 gene of Pchia ciferi ATCC 14091:

Primer CYH1: 5'-CGC GTA GTT AAY GTN CCN AAR AC-3 ',

Primer CYH4: 5'-GCC TGG CCY TTY TGY TTY TTN TC-3 'was synthesized and subjected to PCR reaction with genomic DNA of Peachia ciperi extracted in Example 1 to isolate the L41 gene fragment of about 300bp.

This fragment was labeled according to the product manual using a DIG-labeling detection kit (boehringer Mannheim) to prepare a probe.

<Example 3> Determination of the base sequence of the L41 gene:

The genomic DNA of the chia ciferi of Example 1 was treated with various restriction enzymes, followed by electrophoresis on a 0.9% agarose gel, and transferred to a Nitran membrane (manufactured by Schleicher & Schuell). 6 h reaction at 42 ° C. in a hybrid solution (5X SSC, 0.1% N-laurylsarcosine, 0.02% SDS, 2% blocking agent, 30% formaldehyde) using the prepared probe The treatment was carried out according to the product manual of Boehringer Mannheim.

After administration of the antibody bound to alkaline phosphatase, alkaline bands were observed by adding BCIP and X-phosphate.

A band was identified at the 1.9 kb position of genomic DNA treated with the restriction enzyme EcoR I, and a 1.9 kb EcoR I DNA fragment was extracted and linked to the Eco R I position of plasmid pBluescript KS + (produced by Stratagene), and transformed into E. coli DH5α. The library was prepared.

Southern blotting was performed on the library to obtain 1.9 kb of gene fragments to prepare plasmid pCYH1.9, and DNA sequences were determined using an automatic sequencer (Model 373A, Applied Biosystem). . The results are shown in SEQ ID NO: 1. DNA sequence of the L41 gene of Peachia ciperi ATCC 14091 was registered as AF 053457 (98.3.7) in GeneBank.

The L41 gene of Peachia ciperi ATCC 14091 was composed of a total of 737 bp including an intron of 419 bp, and the 56th amino acid was identified as proline.

<Example 4> Preparation of the plasmid pCYH1.9 ^{r in which} the amino acid No. 56 of the L41 gene is substituted with glutamine:

Primer CH-f: GGT CAA ACC AAA CCA GTT TTC,

Primer CH-r: synthesizes ATG GAA AAC TTG TTT GGT TTG ACC,

The combination of the universal primer and the primer CH-r and the combination of the reverse primer and the primer CH-f were carried out by PCR using pfu DNA polymerase as a template for pCYH1.9 to 1.2kb and A PCR product of 0.7 kb size was obtained.

Both PCR products were subjected to PCR again using only universal primers and regression primers as templates.

In the above, plasmid pCYH1.9 ^r was prepared in which the proline amino acid 56 in the L41 gene was substituted with glutamine.

Example 5 Preparation of Plasmid prDX9.0 Containing Ribosome DNA Fragments

Primer 18R: 5'-CAA TAA TTG CAA TGC TCT ATC CCC AGC ACG-3 ',

Primer 26F: 5'-GGA TAT GGA TTC TTC ACG GTA ACG TAA CTG-3 'was synthesized and subjected to PCR reaction with genomic DNA of Peachiaferi extracted in Example 1 to isolate a 6.0 kb PCR product. The product was labeled by the method of Example 2 to prepare a probe.

When Southern blot with genomic DNA of Peachia ciperi, a band was identified at the 9kb position treated with restriction enzyme XhoI. DNA was extracted from the band position and inserted into the plasmid pBluescript KS + to make a library. Southern blot was performed again to select plasmid prDX9.0 containing a 9 kb ribosomal DNA fragment.

DNA sequence was determined partially using an automatic sequencer, the results of the position and orientation of 26S, 18S, 5.8S, 5S ribosomal genes are shown in FIG.

<Example 6> Preparation of plasmid prXHNC:

3.5kb ribosomal DNA obtained by treating plasmid prDX9.0 of Example 5 with restriction enzyme XbaI was transferred to plasmid pBluescript KS +, and then treated with restriction enzyme HpaI / NcoI / Klenow to remove 1.6kb of ribosomal DNA. Plasmid prXHNC was prepared by inserting 1.9 kb of CYH ^r gene obtained by treating plasmid pCYH1.9 ^r of Example 4 with restriction enzyme EcoRI / Klenow.

<Example 7> Preparation of plasmid prEHC:

The 3.8 kb ribosomal DNA obtained by treating the plasmid prDX9.0 of Example 5 with the restriction enzyme EcoRI was transferred to the plasmid pBluescript KS +, and then treated with the restriction enzyme Hpa I again to remove 2 kb of ribosomal DNA, and the plasmid of Example 4 Plasmid prEHC was prepared by inserting 1.9 kb of CYH ^r gene obtained by treating pCYH1.9 ^r with restriction enzyme EcoRI / Klenow.

<Example 8> Preparation of plasmid prCEX:

1.9 kb of CYH obtained by treating the plasmid pCYH1.9 ^r of Example 4 with the restriction enzyme EcoRI at the EcoRI position of the 1.1 kb ribosome DNA obtained by treating the plasmid prDX9.0 of Example 5 with the restriction enzyme EcoRI / XbaI. ^r gene was combined to make plasmid prCEX.

<Example 9> Preparation of plasmid prCRX:

1.9kb obtained by treating the plasmid pCYH1.9 ^r of Example 4 with the restriction enzyme EcoRI / Klenow at the EcoRV position of the 1.3 kb ribosomal DNA obtained by treating the plasmid prDX9.0 of Example 5 with the restriction enzyme EcoRV / XbaI. The plasmid prCRX was produced by combining the CYH ^r genes.

<Example 10> Preparation of plasmid prXCH:

1.9kb of CYH obtained by treating the plasmid pCYH1.9 ^r of Example 4 with the restriction enzyme EcoRI / Klenow at the HpaI position of the 3.5kb ribosomal DNA obtained by treating the plasmid prDX9.0 of Example 5 with restriction enzyme XbaI. ^r gene was inserted to make plasmid prXCH.

<Example 11> Preparation of plasmid prXCE:

1.9 kb of CYH ^r gene obtained by treating plasmid pCYH1.9 ^r of Example 4 with restriction enzyme EcoRI at the EcoRI position of 3.5 kb ribosomal DNA obtained by treatment of plasmid prDX9.0 of Example 5 with restriction enzyme XbaI. Was inserted to prepare plasmid prXCE.

<Example 12> Preparation of plasmid prXHC1.9:

After plasmid containing 1.6kb ribosomal DNA obtained by treating plasmid prDX9.0 of Example 5 with restriction enzyme XhoI / HindIII, HindIII / Klenow treatment, the plasmid pCYH1 of Example 4 was placed at the EcoRI position of ribosomal DNA. Plasmid prXHC1.9 was prepared by inserting 1.9 kb of CYH ^r gene obtained by treating .9 ^r with the restriction enzyme EcoRI.

<Example 13> Preparation of the plasmid prAC1.9:

1.9kb obtained by treating the plasmid pCYH1.9 ^r of Example 4 with the restriction enzyme EcoRI / Klenow at the EcoRV position of 0.6 kb ribosomal DNA obtained by treating the plasmid prDX9.0 of Example 5 with restriction enzyme HindIII / EcoRV. The plasmid prAC1.9 was produced by combining the CYH ^r genes.

<Example 14> Preparation of the plasmid prEC1.9:

1.9 kb of CYH obtained by treating the plasmid pCYH1.9 ^r of Example 4 with the restriction enzyme EcoRI at the EcoRI position of the 1.4 kb ribosomal DNA obtained by treating the plasmid prDX9.0 of Example 5 with the restriction enzyme HindIII / EcoRI. ^r gene was combined to make plasmid prEC1.9.

<Example 15> Preparation of plasmid prHEC1.9F:

1.9kb obtained by treating the plasmid pCYH1.9 ^r of Example 4 with the restriction enzyme EcoRI / Klenow at the EcoRV position of the 1.4kb ribosomal DNA obtained by treating the plasmid prDX9.0 of Example 5 with the restriction enzyme HindIII / EcoRI. The CYH ^r gene of was inserted to prepare plasmid prHEC1.9F.

<Example 16> Preparation of plasmid prHEC1.9R:

A plasmid prHEC1.9R was prepared in the same manner as in Example 15 except that the CYH ^r was inserted in the opposite direction.

Example 17 Comparison of Transformation Efficiency of Plasmids

According to the class and Peter's method (Klass & Peter, Curr. Genet., 25, 305, 1994), centrifugation was carried out by centrifugation of Peachia peri KFCC-10937 incubated at 100 nm of absorbance at 600 nm in 100 ml of YEPD medium. Then, it was dispersed for 15 minutes at 37 ° C. in 40 ml of 50 mM phosphate buffer (pH7.5) to which 25 mM DTT was added. After washing twice with 100 ml of an ice-cold stabilizer (270 mM sucrose, 10 mM Tris-Cl (pH 7.5), 1 mM MgCl ₂ ), the suspension was suspended in 1 ml of the stabilizer.

5 µl of each plasmid solution (0.1 µg / µl TE) prepared in Examples 6 to 16 was added to 50 µl of the suspension, and left on ice for 10 minutes, followed by a 0.2 mm cuvette for electroporation. Bio-Rad Co.). Using Gean-pulser II (manufactured by Bio-Rad), electrical stimulation was applied under conditions of a voltage of 500 kV, a capacitance of 50 kV and a resistance of 800 kV, and then suspended in 0.5 ml of a stabilizer. 2 ml of YEPD medium was added thereto, followed by incubation at 25 ° C. for 5 hours, then plated on YEPD solid medium to which 10 μg / ml of cycloheximide was added, followed by incubation at 25 ° C. for 4 to 5 days. The number was measured.

Transformation rate is shown in Table 2.

Transformation efficiency by plasmid Plasmid Number of transformants per μg Plasmid Number of transformants per μg prXHNC 276 prEHC 250 prCEX 194 prCRX 226 prXCH 314 prXCE 134 prAC1.9 1574 prXHC1.9 1287 prEC1.9 983 prHEC1.9F 1760 prHEC1.9R 54

From the results of Table 2, it was confirmed that the position of the ribosome DNA into which the CYH ^r gene was inserted and the transcription direction of the CYH ^r gene were closely related to the transformation efficiency, and the non-transcribed region between the 5S and 26S ribosomal RNA structural genes was introduced into the introduction site. In the plasmids prAC1.9 and prHEC1.9F, the transformation efficiency was the highest. In addition, when the transcriptional direction of the 5S RNA gene and the transcriptional direction of the CYH ^r gene were reversed as in the plasmid prHEC1.9R, the transformation efficiency was significantly reduced.

The plasmid prHEC1.9F was selected above.

<Example 18> Transformation conditions setting:

In Example 17, the plasmid prHEC1.9F fixed in capacitance and ring-opened with restriction enzymes Apa I / Sca I was subjected to a voltage of 500, 600, and 700 mA and resistance of 100, 200, 300, 400, 500, 600, and 700. Transformation rate was measured while controlling to 800 kPa.

The results are shown in Table 3.

Transformation conditions Voltage (Ｖ) Resistance Number of transformants per μg 500 100 0 200 82 300 1065 400 1770 500 2890 600 4250 700 6300 800 6850 600 100 0 200 210 300 1488 400 2644 500 5250 600 6750 700 6500 800 4740 700 100 18 200 620 300 2360 400 4400 500 3840 600 2640

From the results in Table 3, it was confirmed that the transformation is most suitable under the conditions of 50 kV capacitance, 500 kV voltage, and 800 kV resistance.

<Example 19> Analysis of transformants by Southern blot:

Each transformant selected in Example 17 was inoculated in YEPD medium to which 5 µg / ml of cycloheximide was added, shaken and cultured at 25 ° C. for 18 to 20 hours, and the cells were collected by centrifugation and transferred to a 1.5 ml tube. . The cells were suspended in 30 µl of STES solution (0.5 M NaCl, 0.01 M EDTA, 1% SDS in 0.2 M Tris-Cl, pH7.6), and then 0.8 volume glass beads of 0.4 mm diameter were added, followed by stirring for 5 minutes. Then, 200 µl of TE buffer (1 mM EDTA in 10 mM Tris-Cl, pH8.0) and 200 µl of phenol / chloroform / isoamyl alcohol (25: 24: 1) were added thereto, followed by stirring for 2 minutes, followed by centrifugation at 12,000 rpm. It was. Genomic DNA was precipitated by adding 2.5 times the volume of ethanol to the supernatant and dried.

2 to 3 µg of genomic DNA was dissolved in 50 µl of distilled water, treated with restriction enzyme EcoRI, and then electrophoresed with 0.8% agarose gel. The band was confirmed by Southern blotting the L41 gene of Example 2 with a probe. It was confirmed that 4 to 5 copies of the L41 gene exist in genomic DNA of all transformants.

<Example 20> Preparation of plasmid prACL2:

(1) Isolation of LCB2 Gene from Peachia Peri ATCC 14091:

The primers L2f and L2r extracted from Saccharomyces cerevisiae cultured in YEPD medium by Johnston's method (Yeast Genetics, molecular aspects, pp. 107-123, IRL Press, 1988):

Primer L2f: 5'-ATG AGT ACT CCT GCA AAC TA-3 '

Primer L2r: 5'-TAA CAA AAT ACT TGT CGT CC-3 '

PCR was performed to isolate the LCB2 gene of Saccharomyces cerevisiae at 1680 bp. Probes were constructed by labeling 913 bp restriction enzyme Sal I fragments using the DIG-labeling detection kit.

The DNA of the chia ciferi of Example 1 was treated with restriction enzymes BamHI, EcoRI, EcoRV, HindIII, PstI, SalI, respectively, and electrophoresed in TAE buffer, followed by Southern blotting with the probe obtained above, BCIP and X-phosphate. Was added to separate the band dyed purple.

After extracting 12kb sized DNA treated with HindIII and linking it to plasmid pBluescript KS +, transformed to E. coli DH5α to prepare a library, and repeated Southern blotting on the library into a 3.0kb ScaI / AflIII fragment. Reduction was made to the plasmid pL2SA.

The base sequence of the LCB2 gene of Peachia ciperi is shown in SEQ ID NO: 2, and registered in GenBank as AF053456 (98.3.7).

The LCB2 gene of Pchia ciferi ATCC 14091 was 1688 bp in size without introns, and was similar in amino acid sequence to that of Saccharomyces cerevisiae.

(2) Construction of the plasmid prACL2:

Plasmid prACL2 was prepared by inserting a 3.0kb LCB2 fragment obtained by treating the plasmid pL2SA obtained in (1) in the order of restriction enzymes HindIII / Klenow / BamHI in the plasmid prHEC1.9F treated with restriction enzyme Eco47III / BamHI.

The plasmid prACL2 prepared above is in the form of ribosomal DNA, CYH ^r (L41), and LCB2 genes.

The plasmid prACL2 obtained above was internationally deposited on 4 May 1998 under the provisions of the Budapest Treaty in the Gene Bank of the Institute of Biotechnology, Korea Advanced Institute of Science and Technology, and was assigned accession number KCTC-0468BP.

<Example 21> Transformation:

The plasmid prACL2, which was opened by treatment with restriction enzyme Apa I, was transformed into the parent strain KFCC-10937 by the method of Example 17, and 10 ² to 10 ³ colonies were formed per µg of plasmid, among which 12 large colonies were selected. It was.

When Southern blot using the L41 gene of Example 2 as a probe, it was confirmed that there are 5 to 10 copies of the gene, one of which was named transformant 1.

<Comparative Example> Production of TAPS of the parent strain KFCC-10937:

The parent strain KFCC-10937 was prepared with YGM optimal medium (glycerol 100g / ℓ, yeast extract 2g / ℓ, KNO ₃ 3g / ℓ, (NH ₄ ) ₂ SO ₄ 0.5g / ℓ, MgSO ₄ · 7H ₂ O 0.3g / ℓ, Inoculated in 100ml of NaCl 0.5g / L, CSL 3g / L, LS-300 1g / L) and incubated at 25 ° C for 4 days at 250rpm, then left at 4 ° C for 2 days, and then chloroform / methanol 1: 1. Four volumes of solvents were added to separate the layers, and then TAPS was extracted.

TAPS was analyzed by HPLC using an electron light scanning detector (ELSD). The dilution ratio of isooctane and THF / formic acid (100: 1.5) was changed to 9: 1, 7: 3, and 9: 1 as solvents.

<Example 22> TAPS production of transformant 1:

Transformant 1 selected in Example 20 was cultured under the conditions of Comparative Example, and TAPS was extracted.

The results of Comparative Example and Example 22 are shown in Table 4.

Comparison of TAPS Productivity between KFCC-10937 and Transformant 1 KFCC-10937 Transformant 1 2 times growth time (hours) 1.5 1.5 Biomass concentration (g / ℓ) 41.6 43.6 TAPS (mg / l) 5206 7444 TAPS specific yield (mg / gdw) 125.1 170.7 Volumetric productivity (mg TAPS / ℓ / hr) 54.2 77.5

When the target gene is transformed into Pchia ciferi using the expression plasmid provided by the present invention, the insertion efficiency of the target gene into the staining agent can be maximized.

<Sequence list>

SEQ ID NO: 1

Sequence length: 1906bp

Type of sequence: nucleic acids and amino acids

Number of chains: 2 prints

Topology: linear

Type of molecule: genomic DNA

origin

Biology: Pichia ciferrii

Main Name: ATCC 14091

Characteristic of the sequence

Characteristic symbols: CDS

Presence Location: 603 .. 605

How to determine the features: E

Characteristic symbols: CDS

Presence Location: 1025 .. 1348

How to determine the features: E

Characteristic symbols: intron

Present Location: 606 .. 1024

How to determine the features: E

SEQ ID NO: 2

Sequence length: 3296bp

Type of sequence: nucleic acids and amino acids

Number of chains: 2 prints

Topology: linear

Type of molecule: genomic DNA

origin

Biology: Pichia ciferrii

Main Name: ATCC 14091

Characteristic of the sequence

Characteristic symbols: CDS

Presence location: 765 .. 2453

How to determine the features: E

Claims (6)

The L41 gene of SEQ ID NO: 1 encoding a ribosomal protein derived from Pichia ciferrii. An expression plasmid for pichia ciferi, comprising a ribosome DNA derived from pichia ciferi, a CYH ^r gene having resistance to the antibiotic cycloheximide, and a target gene. 3. The plasmid according to claim 2, wherein the ribosome DNA derived from the peach ciferi is a non-transcribed site comprising a 1.4kb size obtained by cleaving with HindIII / EcoRI. The plasmid of claim 2 or 3, wherein the target gene is an LCB2 gene as set forth in SEQ ID NO: 2, which encodes a serine palmitoyl transferase derived from pichia ciferi (KCTC-0468BP). ). A method of transforming a target gene into Pchia ciferi using the expression plasmid according to claim 2. A method of extracting tetraacetyl phytosphingosine from the culture medium by culturing the transformed chiasis with the expression plasmid (KCTC-0468BP) according to claim 4, wherein tetraacetyl phytosphingosine is extracted. Production method.