KR20000014502A - Glyceraldehyde-3-phosphate dehydrogenase promoter gene derived from pichia ciferrii and a process for manufacturing taps using thereof - Google Patents

Abstract

PURPOSE: Glyceraldehyde-3-phsphate dehydrogenase (GAPDH) promoter gene isolated from GAPDH gene coding glyceraldehyde-3-phsphate dehydrogenase derived from pichia ciferrii is able to increase yield of tetraacetyl phytosphingosine(TAPS) by amplifying LCB2 gene. CONSTITUTION: Tetraacetyl phytosphingosine(TAPS) is produced by cultivating transformant pichia ciferrii transformed with plasmid containing ribosome DNA derived from pichia ciferrii, CYH gene with resistance to antibiotic cycloheximide, glyceraldehyde-3-phsphate dehydrogenase(GAPDH) promoter gene, LCB2 gene coding serine palmitoyl transferase derived from pichia ciferrii and by extracting tetraacetyl phytosphingosine from culture media.

Description

Glyceraldehyde-3-phosphate dehydrogenase promoter gene derived from peach ciferi and method for producing TAPS using the same

The present invention is a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter gene derived from Pichia ciferrii ATCC 14091, plasmid prACGL2 (KCTC-0498BP) comprising the promoter gene. And prHECGL2 (KCTC-0511BP), and transformed by the chia ciferi transformant and a method for producing a large amount of tetraacetyl phytosphingosine (TAPS: tetraacetyl phytosphingosine) by culturing it.

Ceramides are bonded to the surface of the skin by the double-chain lamellar structure in the stratum corneum to keep the skin smooth and elastic, and it is used in functional cosmetics as it protects the skin and prevents skin drying. come. Its structure is linked to hydroxylated secondary amines with higher fatty acids linked by amide bonds, and these compounds are collectively called ceramides and belong to sphingolipids. {European Patent-B-097,059 number}.

Sphingolipids, including ceramides, are present in the plasma membrane of nucleated cells, and in animals, have functions to regulate cell-cell recognition, cell growth or differentiation. Sphingosine, sphingosine-1-phosphate, lysphingolipids, and ceramides, which are decomposed products of sphingolipids, act as secondary messengers in the signal transduction system {Nagiec et al., Proc. Natl. Acad. Sci. USA, 91, 7899, 1994}.

As described above, although ceramides have various physiological activities, they are present in extremely small amounts in natural plants and animals and microorganisms, and when they are supplied by extraction, the economic burden is very high.

On the other hand, phytosphingosine, including TAPS, not only shows effective effects such as skin protection and skin drying prevention, like ceramides, but is also produced by various microorganisms to facilitate natural raw materials. It is also useful as a raw material of ceramide because it is easily converted to ceramide by N-acylation.

In particular, peach ciferi produces TAPS, which is a phytosphingosine, and has a characteristic of being secreted out of cells, and thus its availability is high {Barenholz et al., Biochem. Biophys. Acta, 248, 458, 1971; ibid, 306, 341, 1973}. 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), which produced The development of mutant strains to increase the number is active {US 5,618,706}. The present inventors have also applied for a patent application by developing a mutant strain (KFCC-10937) having a significantly improved productivity due to an increase in TAPS production and a shortened fermentation period (Korean Patent Application No. 96-67997).

Subsequently, the present inventors have approached genetic engineering to further increase the TAPS productivity of the developed Pchia ciferi KFCC-10937. As a result, the biosynthesis of phytosphingosine including TAPS from wild strain Pchia ciferi ATCC 14091. Cloning the gene LCB2 encoding the serine palmitoyl transferase involved in (SEQ ID NO: 1) and transforming the gene into variant KFCC-10937, resulting in increased TAPS productivity of the transformants (minimum compared to parent strain KFCC-10937). 1.3 times the TAPS production capacity) and confirmed that the plasmid prACL2 (KCTC-0468BP) comprising the gene LCB2 encoding serine palmitoyl transferase derived from Pichia ciferi and thereby transformed with improved TAPS production capacity Transformant "has been applied for a patent {Korean Patent Application No. 98-16309).

Furthermore, the inventors of the present invention, while continuing to study to provide a more improved Peachia Cipari TAPS production capacity, cloned the GAPDH promoter gene of Peachia Ciperi, when using this amplified the expression of the LCB2 gene yield of TAPS It is expected that this can be increased to complete the present invention.

Accordingly, it is an object of the present invention to provide genetic information for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and GAPDH promoters derived from pichia ciferi.

Another object of the present invention is a plasmid prACGL2 and prHECGL2 comprising a GAPDH promoter gene derived from pichia ciferi, and a gene encoding serine palmitoyl transferase derived from pichia ciferi, and thereby transformed to improve TAPS production ability. It is to provide a transformant having.

It is still another object of the present invention to provide a method for mass production of TAPS from the transformed pichia ciferi.

Figure 1 is a restriction map of the GAPDH gene derived from chia ciferi, the direction and length of the arrow indicates the direction of nucleotide sequence determination and the degree of nucleotide sequence determination.

2 is a method for constructing plasmid prACGL2 and a restriction map.

3 is a method for constructing plasmid prHECGL2 and a restriction map.

4 is a Southern blot result for confirming the copy number of the LCB2 gene inserted in the transformant.

In order to achieve the above object, the present invention is to determine the nucleotide sequence of the entire gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the wild strain Pchia siferi ATCC 14091, GAPDH promoter of 600bp size Gene was isolated.

1. Isolation of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH):

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential enzyme involved in glycolysis. It is converted into (1,3-bis-phosphoglycerate) and is always expressed in vivo. Since the GAPDH promoter is not affected by the expression induction process by various carbon sources, active research is being conducted in the field of genetic recombination in order to utilize these characteristics {Kniskern et al., Gene, 46, 135, 1986; Travis et al., J. Biol. Chem., 260, 4384, 1985; Hallewell et al., Biotechnol., 5, 363, 1987; Rosenberg et al., Method Enzymol., 185, 341, 1990; Waterham et al., Gene, 16, 37, 1997}.

Based on these studies, the present inventors also cloned the GAPDH promoter gene of pichia ciferi, and the serine palmitoyl transferase involved in the biosynthesis of phytosphingosine including TAPS of pichia ciferri by this GAPDH promoter. It was confirmed whether the expression of the encoding gene LCB2 was amplified.

The present inventors cloned the GAPDH gene from the genomic DNA of Peachia ciperi ATCC 14091 using GAPDH gene from Saccharomyces cerevisiae 2805 to produce plasmid pGH2.2. The base sequence of the GAPDH gene from Peachia ciperie ATCC 14091 was determined, and the result is shown in SEQ ID NO: 2. The DNA sequence was registered on GenBank as March 05, 1998 as AF053300.

Comparing the nucleotide sequences, the GAPDH gene of Peachia ciperi ATCC 14091 was 1004 bp in size, without introns, and 69.3% identical to the nucleotide sequence of Saccharomyces cerevisiae. The sequence was also 76.2% identical to the amino acid sequence of Saccharomyces cerevisiae. It can be seen that the GAPDH gene sequence of the chia ciferi ATCC 14091 is novel.

2. Isolation of the GAPDH Promoter Gene:

With plasmid pGH2.2, primers:

Primer 3 containing restriction enzyme EcoRV recognition site 5'-GAT ATC TAC ATA CAA TTG ACC CAT AG-3 ' Primer 4 containing restriction enzyme BamHI recognition site 5'-GGA TCC TTA ATT ATT TGT TTG TTT-3 '

The PCR reaction was carried out to isolate the GAPDH promoter gene of 600 bp pitchia ciferi, was inserted into the EcoRV position of pT7-Blue T-vector to prepare a plasmid pT7GH.

3. Isolation of LCB2 gene from which its own promoter was removed:

The plasmid pGAL2 was prepared by separating the LCB2 gene from the plasmid pL2SA {Korean Patent Application No. 98-16309) without the 2.3 kb promoter and inserting it into the BamHI position of the plasmid pT7GH.

4. Construction of Expression Vectors:

A 2.9 kb LCB2 gene was isolated from plasmid pGAL2 and inserted into plasmid prHEC1.9F (Korean Patent Application No. 98-16309) to prepare plasmid prACGL2. This plasmid is linked to the order of ribosome DNA derived from piacyferi, CYH ^r gene (L41), GAPDH promoter gene and LCB2 gene, which are resistant to the antibiotic cycloheximide. Has a restriction map as described. This plasmid was deposited internationally on June 25, 1998 to the Gene Bank of the Korea Institute of Science and Technology, under the provisions of the Budapest Treaty, and was assigned accession number KCTC-0498BP.

On the other hand, when transforming using the plasmid prACGL2, the transformant is transformed by processing a ring enzyme by cleaving one of ribosomal DNA and then transforming the transformant. Inserted together, the insertion of foreign genes cannot be ruled out. The plasmid prHECGL2 was prepared in the form of further comprising an 800 bp ribosomal DNA fragment after the LCB2 gene of plasmid prACGL2. Plasmid prHECGL2 is linked to the sequence of ribosomal DNA derived from pichia ciferi, CYH ^r gene (L41), GAPDH promoter gene, LCB2 gene, and ribosomal DNA derived from pichia ciferi, which are resistant to antibiotic cycloheximide. And the restriction enzyme map shown in FIG. 3. The plasmid was deposited internationally on August 10, 1998 to the Gene Bank of the Korea Institute of Science and Technology, under the provisions of the Budapest Treaty, and was assigned accession number KCTC-0511BP. When transforming with the plasmid prHECGL2, foreign genes can be prevented from being inserted into the chromosome of the transformant by treating restriction enzymes to cut one of each of both ribosome DNAs.

5. Transformation:

Plasmids prACGL2 and prHECGL2 were subjected to electroporation under the conditions of a voltage of 500 mA, a capacitance of 50 mA, and a resistance of 800 mA, and transformed into the production strain Pchia ciferi KFCC-10937, and the plasmid prACL2 (KCTC-0468BP) { Transformant was selected using Korean Patent Application No. 98-16309} as a control.

6. 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 produce TAPS.

Transformants transformed by the plasmids prACGL2 (KCTC-0498BP) and prHECGL2 (KCTC-0511BP) provided by the present invention are at least 2.1 times the control strain plasmid prACL2 (KCTC-0468BP) compared to the parent strain KFCC-10937. It has a TAPS production capacity of at least 1.5 times as compared to the transformant transformed by.

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

Example 1 Preparation of a Probe for Cloning GAPDH Gene

Primer 1: 5'-ATG GTT AGA GTT GCT ATT AAC G-3 '

Primer 2: 5'-AAG CCT TGG CAA TGT GTT CAA-3 'was synthesized and PCR was performed to isolate a 1kb size GAPDH gene from 2805 (available from NIH RBWickner) in Saccharomyces cerevisiae, The plasmid pT7-SGH was prepared by inserting at the EcoRV position of the plasmid pT7-Blue T-vector (manufactured by Novagen).

The plasmid pT7-SGH was digested with restriction enzymes Xba I and Sal I and the 0.9 kb gene fragment was labeled according to the product manual using a DIG-labeling detection kit (boehringer Mannheim) to produce a probe.

Example 2 Extraction of Genomic DNA of Peachia Peri ATCC 14091:

Johnston's method {Yeast Genetics, molecular aspects, pp. 107-123, IRL Press, 1988}, genomic DNA was extracted from Peachia Peri ATCC 14091 incubated in YEPD medium (2% peptone, 1% yeast extract, 2% glucose).

Example 3 Isolation of GAPDH Gene of Pichia Ciferi

The DNA of Example 2 was treated with restriction enzymes BamHI, EcoRI, EcoRV, HindIII, PstI, SalI, respectively, and electrophoresed on 0.9% agarose gel, followed by a Nytran membrane (manufactured by Nytran membrane, Schleicher & Schuell). Then, the probe produced in Example 1 and Southern blot were carried out.

Southern blot conditions were 6 hours at 42 ° C. using a hybrid solution (5 × SSC, 0.1% N-laurylsarcosine, 0.02% SDS, 2% blocking agent, 30% formamide). The reaction 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. The DNA band of about 6 kb treated with HindIII / EcoR I showed positive reaction.

DNA was extracted from the band and linked to plasmid pBluescript KS +, and transformed into E. coli DH5α to prepare a library. Repeated Southern blot was used to select 6.0kb plasmid pGH6.0. In addition, the plasmid pGH2.2 was prepared by reducing the size of the AflIII / HindIII fragment to 2.2 kb.

Restriction map and nucleotide sequence determination method of the GAPDH gene is shown in Figure 1, the results are shown in SEQ ID NO: 2. The nucleotide sequence of GAPDH gene of peach ciferi was registered in GenBank as AF053300 (98.3.7).

GAPDH gene of Peachia ciperi ATCC 14091 is 1004bp without intron and is 69.3% identical to that of Saccharomyces cerevisiae, and the amino acid sequence of Saccharomyces cerevisiae 76.2% were in agreement.

Example 4 Isolation of GAPDH Promoter Gene of Pichia Ciferi:

Primer 3: 5'-GAT ATC TAC ATA CAA TTG ACC CAT AG-3 ',

Primer 4: 5'-GGA TCC TTA ATT ATT TGT TTG TTT-3 'was synthesized and subjected to PCR reaction with the plasmid pGH2.2 prepared in Example 3 to isolate the GAPDH promoter gene of 600 bp pichia ciferi, The plasmid pT7GH was constructed by inserting into the EcoRV position of pT7-Blue T-vector.

<Example 5> Isolation of LCB2 gene from which its own promoter was removed:

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

Primer L2r: 5'-TAA CAA AAT ACT TGT CGT CC-3 'was synthesized and PCR was performed to isolate the LCB2 gene in 1680 bp Saccharomyces cerevisiae. A 913 bp restriction enzyme Sal I fragment was labeled according to the product manual using a DIG-labeling detection kit (product of Boehringer Mannheim) to prepare a probe.

The DNA of Example 2 was treated with restriction enzymes BamHI, EcoRI, EcoRV, HindIII, PstI, SalI, respectively, and electrophoresed on 0.9% agarose gel, followed by a Nytran membrane (manufactured by Nytran membrane, Schleicher & Schuell). It moved and Southern blot with the probe produced above. Southern blot conditions were reacted at 42 ° C. for 6 hours using a hybrid solution (5X SSC, 0.1% N-laurylsarcosine, 0.02% SDS, 2% blocking agent, 30% formamide). 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. Positive reaction was observed in 12 kb sized DNA treated with HindIII.

DNA was extracted from the band and linked to the plasmid pBluescript KS +, transformed into E. coli DH5α to prepare a library, and the Southern blot was repeated for the library to be reduced to a 3.0kb ScaⅠ / AflIII fragment. Plasmid pL2SA was constructed.

Plasmid pL2SA was treated with restriction enzyme AflIII to isolate the LCB2 gene without the promoter of 2.3 kb size, treated with Klenow, and then transferred to plasmid pBluesript KS + treated with BamHI / Klenow to prepare plasmid pL2B2.3.

Plasmid pL2B2.3 was again treated with BamHI to isolate an LCB2 gene of 2.3 kb in size, and was inserted at the BamHI position of plasmid pT7GH of Example 4 to prepare plasmid pGAL2.

Example 6 Preparation of Expression Vector:

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 Peachiaferi extracted in Example 2 to isolate the L41 gene fragment of about 300bp, this PCR product Probe was prepared by labeling by the method of Example 1.

When blotting with genomic DNA of Pchia ciferi, a band was identified at the position of 1.9 kb of genomic DNA treated with restriction enzyme EcoR I, and the 1.9 kb EcoR I DNA fragment was extracted and inserted into plasmid pBluescript KS +. 9 was produced.

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

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 2 to isolate a 6.0 kb PCR product. The product was labeled by the method of Example 1 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.

Plasmid prHEC1 was linked to the 1.9 kb CYH ^r gene obtained by treatment of the plasmid pCYH1.9 ^r with the restriction enzyme EcoRI / Klenow to the EcoRV position of 1.4 kb ribosome DNA obtained by treatment of the plasmid prDX9.0 with the restriction enzyme HindIII / EcoRⅠ. .9F was produced.

The plasmid pGAL2 prepared in Example 5 was treated with restriction enzymes EcoRV and XbaI to isolate a 2.9 kb GAPDH promoter / LCB2 gene. The plasmid prACGL2 was inserted into plasmid prHEC1.9F, which was opened by treatment with restriction enzymes Eco47III and XbaI. Produced.

The plasmid prACGL2 produced in the above form is connected in the order of ribosomal DNA, CYH ^r gene (L41), GAPDH promoter gene and LCB2 gene.

The plasmid prACGL2 was deposited internationally on June 25, 1998 to the Gene Bank of the Korea Institute of Science and Technology, under the provisions of the Treaty of Budapest, and was assigned accession number KCTC-0498BP.

In addition, a 2.9 kb LCB2 gene obtained by treating the plasmid pGAL2 prepared in Example 5 with the restriction enzymes EcoRV and XbaI was inserted into a plasmid pCYH1.9 ^{r that} was opened by treatment with the restriction enzymes Eco47III and XbaI to prepare plasmid pCHGL2. It was. This plasmid was treated with the restriction enzyme EcoRV / XbaI / Klenow, and the 4.6kb sized CYH ^r / GAPDH promoter / LCB2 gene was treated with plasmid prDX9.0 with the restriction enzyme HindIII / EcoRⅠ. Insertion into position produced the plasmid prHECGL2.

The plasmid prHECGL2 produced above is a form further including 800 bp ribosomal DNA after the LCB2 gene of plasmid prACGL2. That is, the plasmid prHECGL2 is in the form of being linked in the order of ribosomal DNA, CYH ^r gene (L41), GAPDH promoter gene, LCB2 gene and ribosomal DNA.

The plasmid prHECGL2 was deposited internationally on August 10, 1998, under the provisions of the Budapest Treaty to the Gene Bank of the Institute of Biotechnology, Korea Research Institute of Science and Technology, and was granted accession number KCTC-0511BP.

The difference between plasmids prACGL2 and prHECGL2 is that during transformation, the plasmid prACGL2 is inserted with the plasmid pBluescript KS + gene (E. coli vector gene) along with the gene from Pchia ciferi, whereas prHECGL2 is inserted only from the Pchia ciferi There is a difference.

Example 7 Transformation:

Classes and Peter's Method {Klass & Peter, Curr. Genet., 25, 305, 1994}, 50 mM phosphoric acid added with 25 mM DTT after centrifugation and recovery of Peachyferi KFCC-10937 incubated in 100 mL of YEPD medium to absorbance at 600 nm. 40 mL of buffer (pH7.5) was dispersed for 15 minutes at 37 ° C. 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.

Plasmid prACGL2 of Example 6 was opened by treatment with restriction enzymes Apa I, plasmid prHECGL2 with restriction enzymes Apa I and Sca I, dissolved in 0.5 µl of 5 µl each, mixed with 50 µl of suspension and left on ice for 10 minutes, 0.2 The electrode was transferred to an electroporation cuvette of mm, manufactured by Bio-Rad. 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. After 2 ml of YEPD medium was added thereto and incubated at 25 ° C. for 5 hours, plated onto YEPD solid medium to which 10 μg / ml of cycloheximide was added and incubated at 25 ° C. for 4 to 5 days. 10 ³ colonies were formed per μg of plasmid, of which 4 large colonies were selected.

Each of the selected transformant colonies 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 enzymes Hind III, EcoR I and Xba I, respectively, and then electrophoresed with 0.8% agarose gel. The bands were confirmed by Southern blotting with L41 gene, LCB2 gene, and GAPDH promoter gene, respectively, labeled with DIG (FIG. 4). About 4-5 copies of the genes were found in the genomic DNA of the transformant. One of the plasmid prACGL2 transformants was selected and named transformant 2, and one of the plasmid prHECGL2 transformants was selected and named transformant 3.

Comparative Example 1 Transformant 1 Transformed by Plasmid prACL2

After opening the plasmid prACL2 by restriction enzyme ApaI, transformant 1 was selected by transforming the parent strain KFCC-10937 by the method of Example 7.

<Comparative Example 2> Production of TAPS of the parent strain KFCC-10937:

The parent strain KFCC-10937 was 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 100g NaCl 0.5g / L, CSL 3g / L, LS-300 1g / L incubated at 250rpm for 4 days at 25 ℃, then left for 2 days at 4 ℃, 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 8 and Comparative Example 3 TAPS Production of Transformants 1 and 2:

Each of transformant 2 selected in Example 7 and transformant 1 selected in Comparative Example 1 were cultured under the conditions of Comparative Example 2, and TAPS was extracted.

The results of Example 8 and Comparative Examples 2 and 3 are shown in Table 1.

Comparison of TAPS Productivity between KFCC-10937 and Transformants 1 and 2 Mother strain KFCC-10937 Transformant 1 (prACL2) Transformant 2 (prACGL2) 2 times growth time (hours) 1.5 1.5 1.5 Biomass concentration (g / ℓ) 41.6 43.6 40.1 TAPS (mg / l) 5206 7444 10933 TAPS specific yield (mg / gdw) 125.1 170.7 272.6 Volumetric productivity (mg TAPS / ℓ / hr) 54.2 77.5 113.9

Example 9 and Comparative Example 4 TAPS Production of Transformant 3:

The transformant 3 selected from Example 7 and the parent strain KFCC-10937 were cultured in YMGL medium {yeast extract 3g / l, malt extract 3g / l, glycerol 30g / l} to extract TAPS under the conditions of Comparative Example 2. .

The results of Example 9 and Comparative Example 4 are shown in Table 2.

Comparison of TAPS Productivity between KFCC-10937 and Transformant 3 Mother strain KFCC-10937 Transformant 3 (prHECGL2) TAPS (mg / ml) 0.200 0.420 TAPS (g / g cell) 0.012 0.027

As can be compared from the results of Table 2, the use of YMGL medium decreased the absolute production compared to the use of YGM optimal medium, but the relative yield of transformant 3 for the parent strain was 2.1 times.

The plasmid prACGL2 provided by the present invention is a 0.6kb ribosome DNA that induces transformation with high efficiency, a CYH ^r (L41) gene, glyceraldehyde-3-phosphate dehydrogen, which is resistant to the antibiotic cycloheximide. The enzyme (GAPDH) promoter gene and the LCB2 gene encoding the serine palmitoyl transferase involved in TAPS biosynthesis are sequentially linked, and the plasmid prHECGL2 further contains 800 bp ribosomal DNA after the LCB2 gene of plasmid prACGL2. In the present invention, the transformation efficiency to the parent strain is high, and the transformants transformed with these plasmids have a TAPS of at least 2.1 times that of the parent strain KFCC-10937 due to the amplification of the LCB2 gene expression by the GAPDH promoter gene. Has production capacity

<Sequence list>

SEQ ID NO: 1

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

SEQ ID NO: 2

Sequence length: 1928bp

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: 907 .. 1911

How to determine the features: E

Characteristic symbols: promoter

Presence Location: 1 .. 906

How to determine the features: E

Claims (11)

The GAPDH gene according to SEQ ID NO: 2 encoding glyceraldehyde-3-phosphate dehydrogenase derived from Pichia ciferrii. The GAPDH promoter gene of SEQ ID NO: 2 isolated from the GAPDH gene of claim 1. Ribosome DNA derived from pichia ciferi, CYH ^r gene resistant to antibiotic cycloheximide, GAPDH promoter gene according to claim 2, and serine palmitoyl transferase derived from pichia ciferi a plasmid comprising an LCB2 gene encoding transferase). The plasmid according to claim 3, which is the plasmid prACGL2 (KCTC-0498BP) having the restriction enzyme map shown in FIG. 4. The plasmid according to claim 3, further comprising ribosomal DNA derived from pichia ciferi of 800 bp after the LCB2 gene. The plasmid according to claim 5, which is plasmid prHECGL2 (KCTC-0511BP) having a restriction map as shown in FIG. The transformation method which can exclude the insertion of the foreign gene containing the vector gene for Escherichia coli which is transfected after opening by processing the plasmid prHECGL2 of Claim 6 with restriction enzymes ApaI and ScaI. Peachia ciperi transformed with the plasmid according to claim 3. Peachia ciperi transformed with the plasmid according to claim 5. A method for producing tetraacetyl phytosphingosine, comprising culturing the transformant pichia ciferi according to claim 8, and extracting tetraacetyl phytosphingosine from the culture. A method for producing tetraacetyl phytosphingosine, comprising culturing the transformant pichia ciferi according to claim 9, and extracting tetraacetyl phytosphingosine from the culture solution.