KR20050081235A - New gene coding for sphinganine hydroxylase and process for the preparation of sphingoid bases - Google Patents

Abstract

The present invention relates to a sphinginine hydroxylase derived from Pichia ciferrii ATCC 14091, a novel SUR2 gene encoding the protein, a recombinant vector comprising the gene, a yeast cell transformed with the recombinant vector, and a phytosphingosine It relates to the production method of the.

Description

New gene coding for sphinganine hydroxylase and method for producing sphingoid base using the gene {New gene coding for sphinganine hydroxylase and process for the preparation of sphingoid bases}

The present invention relates to a sphinganine hydroxylase present in yeast, and more particularly, to a PcSUR2 gene encoding sphingin kinase derived from Pichia ciferrii ATCC 14091.

Sphingolipids are components of the cell membranes of animal cells and are widely distributed in plants and microorganisms (Chem Phys Lipids 5, 6-43, 1970). In addition to the structural functions constituting the cell membrane, sphingolipids also act as anchors of secretory proteins and metabolize sphingolipids such as ceramide, sphingosine, and sphingosine-1-P. Because products act as secondary signaling agents involved in cell growth, death, differentiation, and response to external stimuli, research is actively being conducted (Science 274, 1855-1859, 1996, FASEB J 10, 1388-1397, 1996, Biochem J 335, 465-480, 1998). In addition, ceramide is the main ingredient constituting the stratum corneum of the skin, so it protects the skin from the outside and prevents the skin from drying out, so it is used as a cosmetic raw material. It is emerging.

Sphingolipids have a structure in which fatty acids of various sizes are bound to sphingoid bases. Sphingoid bases that form the backbone of sphingolipids include sphinganine and sphingosine phytosphingosine. Summarizing the biosynthesis of sphingolipids (FIG. 6), 3-ketosphinganine produced by the condensation reaction of amino acids serine (palminetoyl-CoA) and sphinganine In higher cells, fatty acid is added to the amine group of sphinginine and converted to dihydroceramide, and then a ceramide is introduced by sequentially introducing a double bond between the 4th and 5th carbons of the sphinginine. Forms (J. Biol. Chem 272, 21128-21136, 1997). Therefore, ceramides of higher cells are using sphingosine as a basic skeleton. On the other hand, in the case of plants and yeast, hydroxyl groups are introduced into the fourth carbon of sphinginine to form phytosphingosine, and fatty acids are added to produce phytoceramide (Adv Lipi Res 26, 253-274, 1993). . Therefore, sphingolipids of yeast and plants are structures that use phytosphingosine as a basic skeleton.

The SUR2 / SYR2 gene (SUR2 and SYR2 are the same genes) that encode sphinganine hydroxylase was first cloned in the yeast Saccharomyces cerevisiae (Microbiology 142, 4770484, 1996., J.) Biol.Chem 272, 29704-29710, 1997). The gene encodes an enzyme that attaches a hydroxyl group to the fourth carbon of sphinginine, a precursor of phytosphingosine, to convert it into phytostingosine. This gene is not essential for cell growth, but the strain lacking this gene is known to develop resistance against the antibiotic syringomycin-E (J. Biol. Chem 273, 11062-11068, 1997). However, further studies are still needed to understand the physiological role of the sphingoid base hydroxylation.

Pichia ciferra is known as biodesulfurization of coal (Stevens et al., US Patent 4,851,350), production of D-alpha-amino acids (Takeichi et al., US Patent 5,068,187), (S) -1-phenyl-1,3- It can be used for the production of propanediol (Ajinomoto, JP 06090789), secondary alcohol production by stereospecifi ketone reduction (Merck, EP-300287), and in particular, a large amount of TAPS (tetraacetyl) used as a precursor of ceramide Because of its ability to secrete phytosphingosine out of cells, it is widely used to produce TAPS. Recently, the present inventors metabolized the expression of the enzyme serine palmitoyl transferase (3-ketosphinganine synthetase, EC 2.3.1.50), which is involved in the first stage of sphingolipid biosynthesis, to increase the productivity of TAPS. Was prepared (Appl Environ Microbiol 69, 812-819, 2003). In addition to TAPS, there is a need for a system for efficiently producing sphingoid bases such as industrially useful sphinginine and phytosphingosine in Pichia yeast strains.

Against this background, we cloned the SUR2 gene (PcSUR2) derived from Pichia ciferai and transformed the Saccharomyces cerevisiae sur2 deficient strain to restore susceptibility to siringomycin-E. By confirming that phytosphingosine is produced, the present invention was completed by uncovering a novel SUR2 gene derived from Pichia ciferai.

The gist of the invention

The present invention relates to a polynucleotide as set forth in (a) a polynucleotide as set forth in SEQ ID NO: 6, (b) a polynucleotide encoding a protein having at least 90% homology with the sequence of SEQ ID NO 6 and having sphinginine hydroxylase activity and (c) sequence It relates to a gene selected from the group consisting of polynucleotides encoding the sphinginine hydroxylase described in number 7.

In addition, the present invention is selected from the group consisting of (a) a sphinganine hydroxylase set forth in SEQ ID NO: 7 and (b) a protein having at least 90% homology with the sequence of SEQ ID NO: 7 and having sphinginine hydroxylase activity. It relates to the protein being.

The present invention also relates to a vector comprising the gene described above.

The present invention also relates to yeast cells transformed with the vector described above.

In addition, the present invention relates to a method for producing sphinginine hydroxylase, comprising the step of culturing the cells described above to induce and accumulate sphinginine hydroxylase expression, and then separating and purifying the sphinginine hydroxylase.

In addition, the present invention relates to a method for producing phytosphingosine, including culturing the cells to accumulate phytosphingosine in the medium, and then separating and purifying phytosphinginine.

The sphingolipids of yeast or plant use phytosphingosine as the base skeleton, and phytosphingosine is produced by the hydroxylation reaction of sphingin. The enzyme responsible for this reaction is sphinginine hydroxylase, which is known to be able to introduce hydroxyl groups into sphinginin, as well as sphinginin, and thus, in sphingolipid biosynthesis of yeast or plants. It is still uncertain whether the reaction of pingganine or the addition of fatty acids occurs first. However, strains lacking sphinginine hydroxylase did not produce phytosphingosine, and using this property, the SUR2 gene was first isolated from Saccharomyces cerevisiae.

The inventors refer to the DNA sequence of SUR2 gene (ScSUR2) to Saccharomyces cerevisiae (Microbiology 142, 4770484, 1996., J. Biol. Chem 272, 29704-29710, 1997). Southern blot under hybridization conditions containing genomic DNA of Peachia ciperai ATCC 14091 and various concentrations of formamide as probes using an open reading frame (ORF) of the ScSUR2 gene amplified by polymerase chain reaction (PCR) However, the band could not be confirmed. However, when the ScSUR2 gene was analyzed using BLAST, a program that finds similar genes from the database of the gene bank (GenBank / EMBL, NIH, USA), Schizosaccharomyces pombe , a cleavage yeast ), A gene similar to the ScSUR2 gene was found. The two genes were compared using ClustalW, a gene sequence comparison program, and the two enzymes showed 38% similarity in amino acid sequence. Five degenerate primers having base sequences of SEQ ID NOS: 1 to 5 were synthesized based on the sequence of the part with high similarity, and PCR was performed using genomic DNA of Pichia ciferai ATCC 14091 as a template. . When PCR was performed using the primers of SEQ ID NOs: 1 and 3, DNA fragments of about 250bp were amplified, and the nucleotide sequence was linked to the pT7blue vector, which showed 66% homology with the ScSUR2 gene. Therefore, it was confirmed that this gene fragment is part of the PcSUR2 gene. In order to secure the entire PcSUR2 gene, a 250bp gene fragment was probed using a DIG labeling kit, and genomic DNA library and Southern blot of Pchia ciferai ATCC 14091 were performed. Southern blot was repeated to select plasmid pKSR4.5 containing 4.5kb EcoRV fragment containing PcSUR2 gene, and the restriction map is shown in FIG. 1. The base sequence of 2.4 kb including the ORF and promoter of the PcSUR2 gene was determined, and the result is shown in SEQ ID NO: 6. The DNA sequence was registered with GenBank as AY151276, and the plasmid was deposited on February 3, 2004, with the accession number KCTC10591BP to KCTC (Korean Collection for Type Cultures), 52, Eeun-dong, Yuseong-gu, Daejeon, Korea.

As a result of analyzing the nucleotide sequences described above, the PcSUR2 gene was 975 bp and there was no intron, and the amino acid sequence (SEQ ID NO: 7) inferred based on the nucleotide sequence was the amino acid sequence of the sphinginine hydroxylase of Saccharomyces cerevisiae. 60% similarity was shown (FIG. 2). It also contains eight histidine amino acids, the diiron binding sites commonly found in lipid desaturases and lipid hydroxylases, and the dilysil motif sequence found in proteins in the endoplasmic reticulum. It was confirmed that the KKTD amino acid is present at the carboxy terminus.

The inventors linked the ScSUR2 gene and PcSUR2 gene to a promoter of glyceraldehyde 3-phosphate dehydrogenase (GDH1) in Saccharomyces cerevisiae to confirm the function of the PcSUR2 gene. An expression vector such as 3 was prepared and expressed in Saccharomyces cerevisiae sur2 deficient strain. The sur2 deficient strain in Saccharomyces cerevisiae was prepared by transforming Saccharomyces cerevisiae inactivated ScSUR2 gene by URA3 gene insertion into the ORF of the ScSUR2 gene. The sur2 deficient strain in Saccharomyces cerevisiae has been reported to be resistant to the antibiotic siringomycin-E (FEMS Microbiol Lett 131, 63-67, 1995) and to Saccharomycin-E. It has been reported that the mutant strains of Myces cerevisiae recover the susceptibility to siringomycin-E when the ScSUR2 gene is expressed (Microbiology 142. 477-484, 1996). Saccharomyces cerevisiae sur2 deficient strains transformed with PcSUR2 and ScSUR2 gene expression vectors, respectively, were plated and cultured in YEPD medium containing 3 μg / ml of siringomycin-E. As shown, the sensitivity to siringomycin-E was restored and confirmed that it did not grow like wild strain. In addition, sur2 deficiency in Saccharomyces cerevisiae that failed to produce phytosphingosine was analyzed by comparing the amount of sphinginine and phytosphingosine produced by the sur2 deficient strain in Saccharomyces cerevisiae and the strain transformed by the SUR2 gene expression vector. The strain was confirmed by HPLC analysis that the sphinginin accumulated when the PcSUR2 gene or ScSUR2 gene is expressed is converted to phytosphingosine. Therefore, it was confirmed that the gene of sphinganine hydroxylase derived from Pichia ciferai provided in the present invention performs the same function as SUR2 gene in Saccharomyces cerevisiae.

Accordingly, the present invention provides, as one embodiment, a polynucleotide set forth in SEQ ID NO: 6.

The polynucleotide of SEQ ID NO: 6 is a PcSUR2 gene encoding a sphinganine hydroxylase derived from Pichia ciferrii ATCC 14091, and in another embodiment, the present invention has homology with the sequence of SEQ ID NO: 6 A polynucleotide encoding a protein having sphinginine hydroxylase activity or a polynucleotide encoding a sphinginine hydroxylase as set forth in SEQ ID NO: 7 is provided.

The term "homology" as used for the polynucleotide gene in the present invention is intended to indicate a degree similar to that of a wild type nucleic acid sequence, and the polynucleotide sequence encoding the sphingin hydroxy protein of the present invention. Gene sequences which may be at least 75%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% identical. Such homology comparisons are performed using a comparison program that is easy to see with the naked eye. Commercially available computer programs can calculate homology between two or more sequences as a percentage.

In another aspect, the present invention provides a sphinginine hydroxylase polypeptide as set forth in SEQ ID NO: 7.

The polypeptide of SEQ ID NO: 7 may have a different amino acid sequence from the natural polypeptide by deletion, insertion or substitution of one or more amino acids. However, such modified polypeptide variants are also included in the present invention as long as they exhibit sphinginine hydroxylase activity and have the ability to synthesize phytosphingosine. Likewise, fragments of the polypeptide of SEQ ID NO: 7 having sphinginine hydroxylase activity are also included in the present invention. Specifically, in the case of substitutional variants, amino acids may be substituted for "conservation" changes having similar structural or chemical properties, for example, to other amino acids of the class to which a particular amino acid belongs, which class of amino acids may include nonpolar (hydrophobic) amino acids ( Examples: alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine, etc., polar neutral amino acids (e.g. glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, etc.), positive charges (basic) Amino acids (e.g. arginine, lysine, histidine, etc.), and negatively charged (acidic) amino acids (e.g. aspartic acid, glutamic acid, etc.), for example, leucine may be substituted with isoleucine. , Variants may include “non-conserved” changes, eg, glycine substituted with tryptophan, such variants are typically cassette mutagenesis or Other techniques well known in the art can be prepared by constructing a DNA encoding a variant by inducing site-directed mutation of a nucleotide in a DNA encoding a sphingin hydroxylase protein and expressing the DNA through recombinant cell culture. Substitutions of amino acids are typically substitutions of a single base, or one or more substitutions that exhibit functionally identical biological activity, and insertions may typically consist of a contiguous sequence of about 1 to 20 amino acids, but more Large insertions may also be possible The range of deletions is typically about 1 to 30 residues, but in some cases larger deletions may also be possible, such as one of the domains may be deleted. Polypeptides are myristylated, phosphorylated, glycosylated, proteolytic cleavage After detoxification, including can include variations.

The sphinginine hydroxylase of the present invention can be prepared by gene expression of appropriate DNA encoding the protein from an organism isolated or genetically engineered from nature.

In another aspect, the present invention provides a polypeptide having homology with the sequence of SEQ ID NO: 7 and having sphinginine hydroxylase activity.

The term "homology" as used for the polypeptide in the present invention is intended to indicate a degree similar to that of the wild type sphinganine hydroxylase, and the amino acid sequence of the sphinganine hydroxylase of SEQ ID NO: 7 of the present invention. It will comprise an amino acid sequence which may be at least 75%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% identical.

In another aspect, the present invention provides a recombinant vector comprising a sphinginine hydroxylase gene (PcSUR2) of pichia ciferai and a host cell transformed with the recombinant vector.

In the present invention, the gene inserted into the recombinant vector is a gene containing a polynucleotide, which encodes a sphinginine hydroxylase as described above. As described above, the present inventors have a gene having a size of 4.5 kb including the PcSUR2 gene. The containing plasmid pKSR4.5 was deposited with KCTC Accession No. KCTC 10591BP dated 2 / 3-2004 in KCTC. The gene inserted into the recombinant vector of the present invention is preferably a gene encoding a polynucleotide set forth in SEQ ID NO: 6 or a polypeptide set forth in SEQ ID NO: 7, and the present invention provides an expression vector for expressing such a gene.

As used herein, an "expression vector" refers to a gene construct comprising an essential regulatory element operably linked to an insert such that when present in a cell of an individual, the insert is expressed. Standard recombinant DNA techniques can be used to prepare and purify such expression vectors. The type of expression vector is not particularly limited as long as it functions to express a desired gene in various host cells of prokaryotic and eukaryotic cells and to produce a desired protein, but has a promoter with strong activity and strong expression ability, Vectors capable of producing large quantities of foreign proteins of a similar form are preferred. The expression vector preferably contains at least a promoter, a start codon, a gene encoding a desired protein, and a stop codon terminator. In addition, DNAs encoding signal peptides, enhancer sequences, non-translated regions on 5 'and 3' sides of a desired gene, selection marker regions, or replicable units may be appropriately included.

The expression vector as described above can be transformed into a host cell, preferably yeast, more preferably Saccharomyces cerevisiae, Pichia ciferai and the like to produce sphinganine hydroxylase therefrom. As a method for introducing into host cells, the calcium phosphate method or the calcium / rubidium chloride method, electrophoresis described in Sambrook, J. et al., Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, 1. 74 (1989) Electroporation, electroinjection, chemical treatment such as PEG, and the like using a gene gun.

By transforming the transformant transformed with the vector containing the PcSUR2 gene of the present invention, a large amount of sphinganine hydroxylase can be produced and isolated. The medium and culture conditions can be appropriately selected and used depending on the host cell. The nutrient medium preferably contains a carbon source inorganic nitrogen source or an organic nitrogen source necessary for the growth of host cells. Examples of the carbon source include glucose, dextran, soluble starch, sucrose, methanol and the like. Examples of the inorganic or organic nitrogen source include ammonium salts, nitrates, amino acids, corn tips, corn steep liquer, peptone, casein, bovine extract, soybean white, and potato extract. Other nutrients, such as sodium chloride, calcium chloride, sodium dihydrogen phosphate and magnesium chloride, vitamins and antibiotics (tetracycline, neomasin, ampicillin, kanamycin) may be included as needed. During the cultivation, conditions such as temperature, pH of the medium, and incubation time are appropriately adjusted to be suitable for cell growth and mass production of proteins.

Specifically, in the present invention, YGpSU was prepared using YGHR vector derived from YEp352, which is a general yeast expression vector having a GDH1 promoter and a GAL7 terminator, to express sphingin hydroxyases, and transformed strains to Saccharomyces cerevisiae. After the conversion was incubated in 30 ℃ YEPD medium conditions (1% yeast extract, 2% bacto peptone, 2% glucose).

Proteins produced by culturing a transformant as described above and overexpressing sphinginine hydroxylase are isolated by conventional biochemical separation techniques or by immunofriendly methods using antibodies to one or another portion of the protein, It can be purified. Another method may be to add a tag to the protein sequence and purify it by affinity methods using antibodies or other materials with appropriately high affinity for such tags (Sambrook et al., Molecular Cloning: A laborarory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989); Deuscher, M., Guide to Protein Purification Methods Enzymology, Vol. 182. Academic Press. Inc., San Diego, CA (1990)). Conventional biochemical separation techniques include treatment with protein precipitants (salting), centrifugation, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography. There are various chromatography such as chromatography and the like, and in general, when used in combination, high purity proteins can be separated.

Thus, as another embodiment, comprising the step of culturing the transformant transformed with the expression vector containing the PcSUR2 gene of the present invention to express the sphingin hydroxylase, and then separating and purifying sphinginine hydroxylase, Provided are methods for producing sphinganine hydroxylase of Pichia ciferai origin.

The present inventors confirmed the production of phytosphingosine by transforming Saccharomyces cerevisiae sur2 deficient strain with the expression vector YGpSU expressing the PcSUR2 gene of the present invention. Specifically, as shown in Table 1, the sur2 deficient strain did not produce phytosphingosine at all, but the YGpSU transformants transformed with the above expression vector produced phytosphingosine.

Thus, as another embodiment, a method for producing phytosphingosine, comprising the step of culturing the transformant transformed with the expression vector comprising the PcSUR2 gene of the present invention to accumulate phytosphingosine and then to separate and purify the phytosphinginine To provide.

In recent years, as a composition for preventing and protecting skin damage, a variety of substances extracted from natural polymers such as copper, vegetable extracts, collagen, natural products such as amino acids, and vitamins are often prepared. Skin lipids contain small amounts of phytosphingosine and sphingosine in addition to ceramides, cholesterol, and free fatty acids, and in the case of compositions containing ceramides, cholesterol, phytosphingosine, etc., in addition to the above substances, the composition of skin lipids is balanced, thereby improving skin. The effect can be great. Sphingolipids, in particular phytosphingosine, are known to have a wide range of skin disease ameliorating effects. In addition, sphingolipids, such as phytosphingosine and ceramide, have been reported to be effective in skin diseases such as keratin hyperkeratosis, dermatitis, pruritus, bacterial infections and acne seen in atopic dermatitis, eczema and psoriasis (Korean Patent Registration No. 10- 0404072). When sphingolipids are used to treat skin diseases, their use is preferred because there are no side effects of the skin even if they are used for a long time instead of the steroid hormones that have been reported. In addition, ceramide, phytosphingosine and the like are known to have anti-inflammatory effects and the like.

Phytosphingosine produced according to the method of the present invention is a skin disease resulting from a decrease in intracellular phytosphingosine such as atopic dermatitis, eczema, keratin hyperkeratosis, dermatitis, pruritus, bacterial infections, acne or wounds, or It may be provided as an active ingredient of a drug for treating or improving cancer or a cosmetic agent for improving skin.

Hereinafter, the present invention will be described in detail with reference to Examples. The following examples illustrate the invention in detail and are not intended to limit the scope of the invention.

Example

Used strains and experimental materials

L3262 ( MATα ura3-52 leu2-3, 112 his4-34 ) strains in Pichia ciferai ATCC 14091 and Saccharomyces cerevisiae were treated with YEPD medium (1% yeast extract, 2% bacto peptone, 2% glucose). Saccharomyces cerevisiae transformants were selected in SD Ura- medium (0.67% yeast nitrogen base without amino acid, 2% glucose, 0.5% casamino acids). E. coli DH5α was used for general genetic manipulation. D-erythro-sphinganine, D-erythro-sphingosine, and phytosphingosine were purchased from Sigma, St. Louis, Mo. Mycin-E was provided by Dr. J. Takemoto Utah State University, Logan, UT.

Genetic engineering and sequencing method

General genetic manipulation was performed according to techniques such as Sambrook (Molecular cloning: a laboratory manual, 2nd ed, 1989), and the gel extraction kit (viogene) was used to recover genes from agarose gel. Genomic DNA was extracted from yeast according to the method used by Holm (Gene 42, 169-193, 1986) and the like, and yeast transformation was performed according to the method described by Gietz (Yeast 11, 355-360, 1995). E. coli transformation was performed using Inoue (Gene 96, 23-28, 1990). Gene sequencing was performed using Applied Biosystems Model 373A. Primers for polymerase chain reaction were synthesized by Genotech (Korea).

Example 1: Isolation of Sphinganine Hydroxylase Gene from Pichia ciferai

The genomic DNA of Pichia ciperai ATCC 14091 was synthesized by synthesizing the primers of SEQ ID NOs: 1 and 2 running in the forward direction and the primers of SEQ ID NOs: 3, 4 and 5 running in the reverse direction, respectively. PCR was performed with the template. Perkin Elmer (USA) 2400 PCR cycler and Takara (Japan) Ex-taq DNA polymerase were used for 30 reactions at 95 ° C for 30 seconds, 50 ° C for 30 seconds and 72 ° C for 1 minute. Was performed. PCR products amplified on 1% agarose gel were analyzed, and 250 bp gene fragments amplified by the combination of SEQ ID NOS: 1 and 3 were recovered from the gel, ligated to pT7blue vector, and analyzed for sequencing. PCR was again performed using primers of SEQ ID NOs: 1 and 3 together with DIG labeling with DIG-dUTP to probe 250 bp gene fragments that were identified as part of the SUR2 gene of Pichia ciperai. Southern blots were performed on the genomic DNA of Pchia siferai ATCC 14091 to isolate the entire PcSUR2 gene. First, genomic DNAs treated with various restriction enzymes were subjected to electrophoresis on 0.9% agarose gel and fractionated by size, and then transferred to a nylon membrane (Schleicher & Schuell, Germany) by capillary transfer. Hybridization was performed at 42 ° C. for at least 6 hours after adding the probe using a standard solution containing 50% formamide (5 × SSC, 0.1% N-lauroylsarcosine, 0.02% SDS, 5% blocking reagent, 50% formamide). Continued. According to the manufacturer's manual (Boehringer-Mannheim, Germany), the membrane was reacted with an antibody against DIG linked to alkaline phosphatase, and then a color reaction was performed by adding NBT and X-phosphate as substrates. A clear brown signal was confirmed at 4.5 kb of genomic DNA treated with restriction enzyme EcoRV. Therefore, in order to prepare a mini-library containing the PcSUR2 gene, genomic DNA of Pchia ciferai ATCC 14091 was treated with EcoRV and fractionated in 0.9% agarose gel. DNA fragments around 4.5 kb were recovered using a gel extraction kit, and connected to a pBluescriptII vector treated with EcoRV and transformed into E. coli DH5α to prepare a mini-library. Approximately 400 plasmids were purified from the mini-library, followed by Southern blot using the same probe to select plasmid pKSR4.5 containing the entire PcSUR2 gene. Selected plasmid pKSR4.5 was deposited with KCTC Accession No. KCTC 10591BP on February 3, 2004 at KCTC.

Example 2: SUR2 gene expression vector construction

In order to prepare an expression vector for expressing the PcSUR2 gene isolated in Example 1 in a strain in Saccharomyces cerevisiae, pKSR4.5 plasmid was prepared using SEQ ID NOs: 8 and 9, which include primers containing restriction enzyme recognition sites. PCR was performed with the template.

The amplified gene fragment contains only ORF without its own promoter and contains restriction enzyme recognition sites at both ends. The amplified 978bp gene fragment was recovered using a gel extraction kit and treated with restriction enzymes BamHI and SalI, and the PcSUR2 gene expression vector was linked to the BamHI and SalI positions of the YGHR vector, a vector derived from the yeast expression vector YEp352. YGpSU was produced. As shown in FIG. 2, the PcSUR2 gene is located between the GDH1 promoter and GAL7 terminator, which induces strong and constitutive expression, and thus high efficiency expression can be expected. ScSUR2 gene was amplified using the primers of SEQ ID NOs: 10 and 11, and the expression vector YGsSU was produced using the same method as the PcSUR2 gene.

Example 3 Expression Analysis of PcSUR2 Gene in Saccharomyces cerevisiae

Expression of the SUR2 gene on the recovery of susceptibility to syringomycin-E and the production of phytosphingosine after transformation of the expression vector YGpSU vector and YGsSU vector produced in Example 2 into a sur2 deficient strain in Saccharomyces cerevisiae The impact was compared and analyzed. Each transformant selected from SD-Ura medium was plated together with wild strain and sur2 deficient strain in Saccharomyces cerevisiae in YEPD solid medium and 3 μg / ml of siringomycin-E in YEPD solid medium. After incubation for 3 days at 30 ℃. As a result, Saccharomyces cerevisiae sur2 deficient strain transformed with YGpSU vector and YGsSU vector showed the same susceptibility to siringomycin-E.

In order to analyze the production of sphinganine and phytosphingosine produced by each strain, it was analyzed according to the method performed by Min (Anal Biochem 303, 167-175, 2002). The yeast cells were recovered from 1 ml culture medium incubated in YEPD liquid medium from 30 ° C. shaker to late logarithmic phase and suspended in 0.1 ml of distilled water. Glass beads were added to an amount that could be submerged in distilled water and then stirred for 5 minutes. 0.2 ml of distilled water was added, followed by centrifugation at 12000 rpm for 10 minutes to recover only the supernatant. To determine the concentration of protein, 20 μl was stored at 4 ° C., and then 10 pmol of C17-sphingosine was added as an internal standard, followed by 0.8 ml of chloroform / methanol (2: 1, v / v) at room temperature. Stir for minutes. The layers were separated by centrifugation at 12000 rpm for 10 minutes, the lower layer was transferred to a new tube, dried, and dissolved in 0.2 ml of chloroform / methanol (9: 1, v / v) solution. 15 μl of o-phthalaldehyde (OPA) solution [50 mg OPA, 1 ml ethanol, 10 ml 2-mercaptoethanol, 50 ml 3% boric acid solution (pH 10.5)] was added and reacted at room temperature for 20 minutes. HPLC analysis was performed using a C18 reversed phase column (Cosmosil, 5C 18-AR-II; 4.6 mm id × 150 mm), flow rate of 1 ml / min, separation solvent was acetonitrile: distilled water (9: 1) Analysis was performed using a Jasco FP-920 spectrofluorometer at 340 nm and emission wavelength 455 nm. The sphingoid base yield analysis of YGpSU or YGsSU transformants is shown in Table 1 below.

Strain Sphinganine (pmol / mg protein) Phytosphingosine (pmol / mg protein) sur2 deletion strain  346.8 0.0 YGpSU transformant 2.6 178.7 YGsSU transformant 2.4 194.1

As shown in Table 1, the sur2 deficient strain in Saccharomyces cerevisiae that failed to produce phytosphingosine was converted to phytosphingosine, which was accumulated when the PcSUR2 gene or ScSUR2 gene was expressed.

Recombinant expression vectors and transformants in which the sphinganine hydroxylase gene derived from Pichia ciferai of the present invention are controlled by the GDH1 promoter provide a system capable of mass-producing sphinganine hydroxylase and phytostingosine and phytosphingosine There is an effect of the invention to provide a broad range of skin improvement compositions comprising sphingolipids, such as de.

1 is a diagram showing a restriction map of pKSR4.5, a plasmid containing SUR2 gene derived from Pichia ciferrii isolated from the present invention.

FIG. 2 is a diagram comparing amino acid sequences of sphinganine hydroxylases derived from Pichia ciferai and Saccharomyces cerevisiae .

3 is a diagram showing a SUR2 gene expression vector.

4 is a diagram showing the susceptibility to siringomycin-E of strains transformed with SUR2 gene expression vector.

5 is a diagram showing the structure of sphingoid base and ceramide.

6 shows the biosynthetic pathway of sphingolipids.

<110> Korea Reserarch Institute of Bioscience and Biotechnology <120> New gene coding for sphinganine hydroxylase and process for the preparation of sphingoid bases <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for the isolation PcSUR2 <400> 1 gaaaaatatm gwatycatcc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for the isolation PcSUR2 <400> 2 tggcaatatt tytkrcatcg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for the isolation PcSUR2 <400> 3 tawccacaat gatcatcrac 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for the isolation PcSUR2 <400> 4 tgatgatgwa watcatgata 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for the isolation PcSUR2 <400> 5 tcccaraawg traaraawgg 20 <210> 6 <211> 2400 <212> DNA <213> Pichia ciferrii <220> <221> CDS <222> (547) .. (1521) <220> <221> terminator (222) (1522) .. (1524) <400> 6 cgtctctttc tccccagtgc gctgaccagt tccctgtcta ttttatgggc agatggcctt 60 gtctaggcag acctaaatag aaagtaggca gagtgcacct gtgaccagcc tgcccggcta 120 gtggcaagtg ccctcttagt gcccgcctaa cccctttctt ttgagcgttg cgcgctgcga 180 agaaaagaaa gaacaagtga aaagtttaaa aacatttcaa acaaacaaat aaatgcacag 240 atcgccataa tttcacgtaa acaagagaga tttaattaaa taaaccttgt taacaaacac 300 aaatcaaaac aagaccatta agtttataaa gataaagttt tgctgggatt tatcacgttt 360 agtacacagc aaaaagtttt aagtactagt tttctagatt ttgacaggta taaaagacca 420 tttttctcgt ttaaaaacat ctcaaaaaca tcacaaggtc cgttgagtgt tatataaaca 480 ttgagaatcc atcaagtttc ttacaacaaa atcttttgat agtttgagat catatacaca 540 aaagac atg agc tct cat cag ttt ttg atc aac caa aca act ttg gcg 588 Met Ser Ser His Gln Phe Leu Ile Asn Gln Thr Thr Leu Ala 1 5 10 gct cca cct gtt cat ttg gtg gag aaa cca agt ttg att aat ggc ata 636 Ala Pro Pro Val His Leu Val Glu Lys Pro Ser Leu Ile Asn Gly Ile 15 20 25 30 ccg gat aac att tta gcc ttg att gca cct gtt ata gct tat tat tca 684 Pro Asp Asn Ile Leu Ala Leu Ile Ala Pro Val Ile Ala Tyr Tyr Ser 35 40 45 tat tca gga ttt ttc tat gtg att gat act tta gaa att gca gaa ctt 732 Tyr Ser Gly Phe Phe Tyr Val Ile Asp Thr Leu Glu Ile Ala Glu Leu 50 55 60 tat aga att cat cca cct gaa gaa gtt agt tca aga aat aaa gct aca 780 Tyr Arg Ile His Pro Pro Glu Glu Val Ser Ser Arg Asn Lys Ala Thr 65 70 75 aaa ttt gat gtt tta aaa gat gtt gtt tta caa cat ttt ata cag agt 828 Lys Phe Asp Val Leu Lys Asp Val Val Leu Gln His Phe Ile Gln Ser 80 85 90 gtt gtt ggt tat atc ttt aca tat ttt gat cca att caa tat act ggt 876 Val Val Gly Tyr Ile Phe Thr Tyr Phe Asp Pro Ile Gln Tyr Thr Gly 95 100 105 110 gat gaa gaa tat caa gct tgg aaa tta caa caa act tta cca ttt tta 924 Asp Glu Glu Tyr Gln Ala Trp Lys Leu Gln Gln Thr Leu Pro Phe Leu 115 120 125 cca ttt gat gtt gca tat tat tgg aat atg tat ggt tgg agt tgt ttg 972 Pro Phe Asp Val Ala Tyr Tyr Trp Asn Met Tyr Gly Trp Ser Cys Leu 130 135 140 aaa att ggt ctt gca ttt tta att att gat tca tgg caa tat tgg tta 1020 Lys Ile Gly Leu Ala Phe Leu Ile Ile Asp Ser Trp Gln Tyr Trp Leu 145 150 155 cat aga att atg cat tta aac aag aca tta tac aaa aga ttc cat tca 1068 His Arg Ile Met His Leu Asn Lys Thr Leu Tyr Lys Arg Phe His Ser 160 165 170 aga cat cat cgt ctt tat gtc cca tat gct ttt ggt gct tta tat aat 1116 Arg His His Arg Leu Tyr Val Pro Tyr Ala Phe Gly Ala Leu Tyr Asn 175 180 185 190 gat cca ttt gaa ggg ttt tta ttg gat acc tta ggt acc ggt att gct 1164 Asp Pro Phe Glu Gly Phe Leu Leu Asp Thr Leu Gly Thr Gly Ile Ala 195 200 205 gca att gtt act caa tta act cca aga gaa tct att gtt tta tat aca 1212 Ala Ile Val Thr Gln Leu Thr Pro Arg Glu Ser Ile Val Leu Tyr Thr 210 215 220 ttt tca act ttg aaa act gtt gat gat cat tgt ggt tat tca tta cct 1260 Phe Ser Thr Leu Lys Thr Val Asp Asp His Cys Gly Tyr Ser Leu Pro 225 230 235 tat gat cct ttc caa att ttg ttc cca aat aac tca att tat cat gat 1308 Tyr Asp Pro Phe Gln Ile Leu Phe Pro Asn Asn Ser Ile Tyr His Asp 240 245 250 att cat cat caa caa ttt ggt atc aag acc aat ttt tca caa cct ttc 1356 Ile His His Gln Gln Phe Gly Ile Lys Thr Asn Phe Ser Gln Pro Phe 255 260 265 270 ttt aca cat tgg gat gtt ttc agt aat aca aga tat aaa gaa att gat 1404 Phe Thr His Trp Asp Val Phe Ser Asn Thr Arg Tyr Lys Glu Ile Asp 275 280 285 gaa tac aga gaa aag caa aaa gct att aca att gcc aaa tat aaa gag 1452 Glu Tyr Arg Glu Lys Gln Lys Ala Ile Thr Ile Ala Lys Tyr Lys Glu 290 295 300 ttt tta cat gat cgt gaa att gca aaa caa aag aag aag gct gaa att 1500 Phe Leu His Asp Arg Glu Ile Ala Lys Gln Lys Lys Lys Ala Glu Ile 305 310 315 tat aaa gat aag aaa act gat tgatttcga tcaaattttc attataaatt 1550 Tyr Lys Asp Lys Lys Thr Asp 320 325 tatgactaat atcattttac aaacaatttt gtcattatac ccaacattat taatctaata 1610 aatcacaatt taacttttgt tgtgttatat atatacattt actttaaaat accctttttg 1670 gaagaaaact aatttaatca cttaatgaat acatgaattt aatttattat aaacacagcg 1730 gctcataatt aagtagtaca attaattgca atattccttg agcttttatc gaaatggaaa 1790 attatatcaa tattcggatg atggttatgt tcttttaata attctattcg atcttgtata 1850 accaatgttt caatgacatg acttaaaaat ccataaacaa aacaccattt aatgaaaatt 1910 ccacaagcat tatgaactga tctgtcttgt tcaacttcaa accttttaat aatctcttga 1970 ttgtctaaag ctcgtagatc tgaagttcca aatactttaa aattaacatc atgctccata 2030 aacttcaaag aaatgaactt tgtaacttta tggaagcaac aaggaacgaa taaacaacca 2090 ataacatcta agaaacttgg acttttgacc acaagatcta tgatgtttcc agataaatca 2150 ccacaagtat ccaatccaat aatcaaacat ttgggtttac cgctattcga tctttgtctt 2210 gatcttatga atctcaacgt atcttcatca ttcaactcaa agtgtttagt ataatgaact 2270 tgattgtcct ccagcgatga tacactatgt ttcattaatc cattgagtct tttattaact 2330 ttttgtgcct tgagattggg ttcaatagct atagtgggaa gtttatgtag tcttgataaa 2390 tttgaattcc 2400 <210> 7 <211> 325 <212> PRT <213> Pichia ciferrii <400> 7 Met Ser Ser His Gln Phe Leu Ile Asn Gln Thr Thr Leu Ala Ala Pro 1 5 10 15 Pro Val His Leu Val Glu Lys Pro Ser Leu Ile Asn Gly Ile Pro Asp 20 25 30 Asn Ile Leu Ala Leu Ile Ala Pro Val Ile Ala Tyr Tyr Ser Tyr Ser 35 40 45 Gly Phe Phe Tyr Val Ile Asp Thr Leu Glu Ile Ala Glu Leu Tyr Arg 50 55 60 Ile His Pro Pro Glu Glu Val Ser Ser Arg Asn Lys Ala Thr Lys Phe 65 70 75 80 Asp Val Leu Lys Asp Val Val Leu Gln His Phe Ile Gln Ser Val Val 85 90 95 Gly Tyr Ile Phe Thr Tyr Phe Asp Pro Ile Gln Tyr Thr Gly Asp Glu 100 105 110 Glu Tyr Gln Ala Trp Lys Leu Gln Gln Thr Leu Pro Phe Leu Pro Phe 115 120 125 Asp Val Ala Tyr Tyr Trp Asn Met Tyr Gly Trp Ser Cys Leu Lys Ile 130 135 140 Gly Leu Ala Phe Leu Ile Ile Asp Ser Trp Gln Tyr Trp Leu His Arg 145 150 155 160 Ile Met His Leu Asn Lys Thr Leu Tyr Lys Arg Phe His Ser Arg His 165 170 175 His Arg Leu Tyr Val Pro Tyr Ala Phe Gly Ala Leu Tyr Asn Asp Pro 180 185 190 Phe Glu Gly Phe Leu Leu Asp Thr Leu Gly Thr Gly Ile Ala Ala Ile 195 200 205 Val Thr Gln Leu Thr Pro Arg Glu Ser Ile Val Leu Tyr Thr Phe Ser 210 215 220 Thr Leu Lys Thr Val Asp Asp His Cys Gly Tyr Ser Leu Pro Tyr Asp 225 230 235 240 Pro Phe Gln Ile Leu Phe Pro Asn Asn Ser Ile Tyr His Asp Ile His 245 250 255 His Gln Gln Phe Gly Ile Lys Thr Asn Phe Ser Gln Pro Phe Phe Thr 260 265 270 His Trp Asp Val Phe Ser Asn Thr Arg Tyr Lys Glu Ile Asp Glu Tyr 275 280 285 Arg Glu Lys Gln Lys Ala Ile Thr Ile Ala Lys Tyr Lys Glu Phe Leu 290 295 300 His Asp Arg Glu Ile Ala Lys Gln Lys Lys Lys Ala Glu Ile Tyr Lys 305 310 315 320 Asp Lys Lys Thr Asp 325 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer for the expression PcSUR2 <400> 8 ggatcctata cacaaaagac atg 23 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer for the expression PcSUR2 <400> 9 gtcgacaatt tgatcgaaat ca 22 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer for the expression ScSUR2 <400> 10 ggattcgaaa agaaaatata cgatg 25 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer for the expression ScSUR2 <400> 11 gtcgacaggt atgcaaaagg gtta 24

Claims (10)

(a) a polynucleotide set forth in SEQ ID NO: 6, (b) a polynucleotide encoding a protein having at least 90% homology with the sequence of SEQ ID NO: 6 and having sphinginine hydroxylase activity, and (c) SEQ ID NO: 7 A gene selected from the group consisting of polynucleotides encoding the sphinginine hydroxylase described. The gene according to claim 1, wherein the gene is a polynucleotide of Genbank Registration No. AY151276, set forth in SEQ ID NO: 6. The gene of claim 1, which is a polynucleotide encoding a sphinginine hydroxylase set forth in SEQ ID NO: 7. A protein selected from the group consisting of (a) a sphinginine hydroxylase set forth in SEQ ID NO: 7 and (b) a protein having at least 90% homology with the sequence of SEQ ID NO: 7 and having sphinginine hydroxylase activity. The sphinganine hydroxylase protein of claim 4, wherein the sphinganine hydroxylase protein is set forth in SEQ ID NO: 7. 6. A vector comprising the gene of claim 1. The pKSR4.5 vector of claim 6, having Accession No. KCTC 10591BP. Yeast cells transformed with the vector of claim 6. The cell of claim 8, wherein the yeast cell is Saccharomyces cerevisiae or Pichia ciferrii . A method of producing phytosphingosine comprising culturing the cells of claim 8 to accumulate phytosphingosine, and then separating and purifying the phytosphingosine.