KR100267668B1 - A gene coding for serine palmitoyl transferase of pichia ciferii and a method for producing taps - Google Patents

Abstract

PURPOSE: A serine palmitoyl transferase coding gene derived from Pichia ciferrii and a tetraacetyl phytosphingosine(TAPS) producing method using the same are provided, thereby tetraacetyl phytosphingosine(TAPS) can be mass produced with low costs. CONSTITUTION: The gene LCB2 encoding serine palmitoyl transferase and derived from Pichia ciferrii (ATCC 14091) is represented by sequence ID No. 1 described as in the description. The expression vector prACL2(KCTC-0468BP) having an endonuclease map of Fig. 3 contains the gene LCB2, CYHr gene being resistant to cycloheximide, and ribosome DNA derived from Pichia ciferrii, wherein the ribosome DNA comprises a 0.6kb non transcribed region which is obtained by cleavage with HindIII/EcoRV. A transformant is produced by transforming Pichia ciferrii with the expression vector prACL2(KCTC-0468BP). Tetraacetyl phytosphingosine(TAPS) which is a raw material of ceramides is produced by incubating the transformed Pichia ciferrii and extracting TAPS from the fermented culture, wherein ceramides are used in protecting the skin and maintaining the humidity of the skin.

Description

Gene encoding serine palmitoyl transferase derived from peach ciferi and method for producing TAPS using the same

The present invention relates to a gene LCB2 encoding serine palmitoyl transferase from Pichia ciferrii ATCC 14091, plasmid prACL2 (KCTC-0468BP) comprising the gene, and blood thus transformed. Tooth ciferi, and a method for culturing the same, a method for mass production of tetraacetyl phytosphingosine (TAPS: tetraacetyl phytosphingosine).

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. In its structure, higher fatty acids are linked to hydroxylated secondary amines by amide bonds, and these compounds are collectively called ceramides and belong to sphingolipids (EP B 097,059).

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 signal transduction systems (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, Pchia ciferi is characterized by producing TAPS, a phytosphingosine, and secreting it out of cells, and thus has high availability (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). The development of mutant strains to increase is active (US Patent 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 No. 96-67997).

Furthermore, the present inventors have taken a genetic engineering approach to further increase the TAPS productivity of the developed Peachia ciferi KFCC-10937. As a result, we cloned a gene encoding serine palmitoyl transferase involved in the biosynthesis of phytosphingosine including TAPS from wild strain Pchia ciferi ATCC 14091, and the mutant strain KFCC-10937 developed by the present inventors. The present invention was completed by confirming the increased TAPS productivity of the transformants.

Accordingly, it is an object of the present invention to provide genetic information for serine palmitoyl transferase derived from pichia ciferi involved in the biosynthesis of TAPS.

Another object of the present invention is to provide a plasmid prACL2 comprising a gene encoding a serine palmitoyl transferase derived from pichia ciferi and a transformant transformed thereby having improved TAPS production capacity.

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 LCB2 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.

Fig. 2 is a map of restriction enzymes of plasmids in which L41 genes derived from pichia ciferi are respectively inserted at various sites of the ribosomal gene.

3 is a method for preparing plasmid prACL2 and a restriction map.

In order to achieve the above object, the present invention clones the gene LCB2 encoding serine palmitoyl transferase involved in the biosynthesis of phytosphingosine including TAPS from wild strain Pchia ciferi ATCC 14091 Decided.

1.Isolation of the gene LCB2 encoding serine palmitoyl transferase:

Looking at the biosynthesis of sphingolipids, the first step is the 3-ketosphinganine synthase (EC 2.3.1.50) condensing serine and palmitoyl co-enzyme (3-mi-ketose with 18 carbon atoms). Forming a 3-ketosphinganine, which is the rate limiting step of the sphingolipid biosynthesis process (Barenholz et al., Biochem. Biophys. Acta, 248, 458, 1971; ibid , 306, 341, 1973). Later, 3-ketosphinginine is used as a long chain in animals and as a precursor of phytosphingosine in plants and fungi.

In other words, the present invention cloned serine palmitoyl transferase, an enzyme acting in the rate determining step, from wild strain Peach ciferi ATCC 14091, which resulted in shortened fermentation period developed by the present inventors and greatly improved productivity. TAPS production capacity of the transformant obtained by the transformation was confirmed.

In order to clone the gene encoding the serine palmitoyl transferase from genomic DNA of Peachia ciperi ATCC 14091, we first constructed a probe with reference to the subunits of the known serine palmitoyl transferase gene.

The gene encoding serine palmitoyl transferase from Saccharomyces cerevisiae consists of two subunits, LCB1 and LCB2 (LCB stands for long chain base; Nagiec et al) , Proc. Natl. Acad. Sci. USA, 91, 7899, 1994), human, mouse, DNA of LCB2 gene from Klebsiella lactis, Schizosaccharomyces pombe Base sequences are also known (Nagiec et al., Gene, 177, 237, 1996; Hanada et al., J. Biol. Chem., 272, 32108, 1997; Weiss & Stoffel, Eur. J. Biochem., 249 , 239, 1997).

First, referring to the DNA sequence of the LCB1 gene derived from Saccharomyces cerevisiae (Nagiec et al., Proc. Natl. Acad. Sci. USA, 91, 7899, 1994), the 1kb size of the LCB1 gene No band was identified when the PstI fragment was Southern blotted with genomic DNA of Peachia ciperie ATCC 14091 with a probe. However, when a 0.9 kb SalI fragment of LCB2 gene was Southern blotted with a probe, a DNA band containing a 12 kb LCB2 gene was identified and inserted into a plasmid to prepare a library. Southern blot was repeated to reduce plasmid pL2SA to 3.0 kb ScaI / AflIII fragments (FIG. 1). The base sequence of the LCB2 gene was determined, and the result is shown in SEQ ID NO: 1. 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.

2. Transformation method:

Peachia ciperi ATCC 14091 was called the genus Hansenula, but was recently renamed as the genus Peach by 5S-RNA analysis (Yamada et al., Biosci. Biotechnol. Biochem., 58, 1245, 1994). Genetic studies on the Peach subspecies have been insufficient, so no method for transforming Peach ciferi strains has been developed. Accordingly, the present inventors have developed a transformation method for chia ciferi separately from the present invention, and only a part of the results are described herein.

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

2-1. Construction of plasmid pCYH1.9 ^r for transformant selection:

The plasmid pCYH1.9 was constructed by cloning the ribosomal protein L41 gene of Peachia ciperi ATCC 14091, and DNA sequencing was determined. The results are shown in SEQ ID NO: 2. DNA sequence was registered in GenBank as AF053457 (98.3.7).

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. Amino acid No. 56, which is sensitive to the antibiotic cycloheximide, was identified as proline.

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

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 .

2-2. Construction of the plasmid prDX9.0 to increase the efficiency of insertion of the target gene into the chromosome:

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

PCR was performed using primers synthesized by referring to the partial sequencing of the peach ciferi ribosomal RNA (Yamada et al., Biosci. Biotechnol. Biochem., 58, 1245, 1994) to obtain a 6.0 kb ribosomal DNA fragment. The fragment was Southern blotted with a probe to separate ribosomal DNA fragment of 9 kb Peachia ciperi ATCC 14091, and inserted into plasmid pBluescript KS + to prepare plasmid prDX9.0.

2-3. Transformation and transformant screening:

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.

3. Construction and transformation of recombinant plasmid prACL2 containing LCB2 gene:

According to the method shown in FIG. 3, a recombinant plasmid prACL2 containing an LCB2 gene was prepared, and the plasmid has a form in which CYH ^r and LCB2 genes are connected to ribosomal DNA fragments side by side. The plasmid was deposited internationally on May 4, 1998, under the provisions of the Budapest Treaty to the Gene Bank within the Institute of Biotechnology, Korea Research Institute of Science and Technology, and was assigned accession number KCTC-0468BP.

This plasmid was transformed into the mutant strain Peach ciferi KFCC-10937 developed by the inventors according to the method described in 2-3. Above, and a transformant having a high gene copy number was selected.

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> Preparation of a probe for cloning LCB2 gene:

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.

<Example 2> Extraction of genomic DNA of Pchia ciferi ATCC 14091:

Genome from Peachiaferri ATCC 14091 incubated 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). DNA was extracted.

<Example 3> Preparation of plasmid pL2SA:

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

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 this library to be reduced to a 3.0kb ScaⅠ / AflIII fragment. Plasmid pL2SA was constructed.

Restriction enzyme map and nucleotide sequence determination method of the LCB2 gene is shown in Figure 1, the results are shown in SEQ ID NO: 1. The base sequence of the LCB2 gene of Peachia ciperi was 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.

<Example 4> Extraction 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 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.

The base sequence for the 1.9 kb size DNA contained in the plasmid pCYH1.9 was determined, and the result is shown in SEQ ID NO: 2. 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 5> 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 6 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 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.

<Example 7> Preparation of plasmids in which the L41 gene was inserted into various sites of ribosomal DNA, respectively:

A fragment obtained by treating the plasmid pCYH1.9 ^r of Example 5 with the restriction enzyme EcoR I was inserted into a plasmid obtained by treating prDX9.0 with various restriction enzymes shown in FIG. The inserted plasmid was prepared.

As an example, plasmid prHEC1.9F is introduced as a property, and the plasmid pCYH1.9 of Example 5 is located at the EcoRV position of 1.4 kb of ribosomal DNA obtained by treating the plasmid prDX9.0 obtained in Example 6 with the restriction enzyme HindIII / EcoRI. ^The plasmid prHEC1.9F was prepared by linking 1.9 kb of CYH ^r gene obtained by treating ^r with restriction enzyme EcoRI.

<Example 8> Selection of the plasmid prHEC1.9F:

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 the various plasmid solutions (0.1 µg / µl TE) prepared in Example 7 were added to 50 µl of the suspension, which was left on ice for 10 minutes, followed by 0.2 mm of Cuvette, Bio- for electroporation. 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. 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 1.

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 1, 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 (non- Transformation efficiency was highest for prHEC1.9F, a plasmid using the transcribed sequence as an introduction site.

Through this process, a plasmid prHEC1.9F containing 1.4kb ribosomal DNA and CYH ^r gene was used to induce transformation with high efficiency.

<Example 9> Preparation of plasmid prACL2:

Plasmid prACL2 was prepared by inserting a 3.0kb LCB2 fragment obtained by treating the plasmid pL2SA of Example 3 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 was linked in the order of 0.6 kb of non-transcribed region of HindIII / EcoRV, CYH ^r (L41), and LCB2 gene of ribosomal DNA.

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 10> Transformation:

After opening the plasmid prACL2 with the restriction enzyme Apa I by the method of Example 8, the strain was transformed into the parent strain KFCC-10937 by the method of Example 8, and 10 ² to 10 ³ colonies were formed per μg of the plasmid, among which Twelve large colonies were selected.

Each of the selected transformants 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 recovered 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 4 with a probe. It was confirmed that 5 to 10 copies of the L41 gene were present in the genomic DNA of the transformant. One of them 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 11 TAPS Production of Transformant 1:

Transformant 1 selected in Example 10 was cultured under the conditions of Comparative Example, and TAPS was extracted.

The results of Comparative Example and Example 11 are shown in Table 2.

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

Plasmid prACL2 provided by the present invention is a sequence of 0.6kb ribosome DNA, which induces transformation with high efficiency, and the LCB2 gene encoding the serine palmitoyl transferase involved in CYH ^r (L41) gene and TAPS biosynthesis. In a linked form, the transformation efficiency to the parent strain is high, and the transformants transformed by this plasmid have a TAPS production capacity of at least 1.3 times that of the parent strain KFCC-10937.

<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: 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

.

Claims (5)

The LCB2 gene set forth in SEQ ID NO: 1 encoding serine palmitoyl transferase from Pichia ciferrii. Ribosome DNA derived from chia ciferi, CYH ^r gene resistant to antibiotic cycloheximide, and LCB2 gene according to claim 1; Plasmid prACL2 (KCTC-0468BP) having a restriction map as shown in FIG. 3. The plasmid prACL2 according to claim 2, wherein the ribosome DNA derived from the chia ciferi comprises a non-transcribed region comprising a size of 0.6 kb obtained by cleaving with HindIII / EcoRV. Peachia ciperi transformed with plasmid prACL2 according to claim 2. A method for producing tetraacetyl phytosphingosine, comprising culturing the transformant pichia ciferi according to claim 4, and extracting tetraacetyl phytosphingosine from the culture.