KR100613753B1 - 77 A gene coding serine palmitoyltransferase of Sphingomonas Chungbukensis DJ77 recombination vector transformation organism using thereby serine palmitoyltransferase protein and the preparation method thereof - Google Patents

Abstract

The present invention relates to a gene encoding serine palmitoyltransferase (SPT) in Sphingomonas Chungbukensis DJ77, specifically, spingolipids biosynthesis process. Provided are a gene encoding SPT, a recombinant vector thereof, a transformant using the same, a method for producing the same, and a method for producing the SPT protein encoded by the gene sequence and a method for producing the same.

Sphingomonas Chungbuk Kensis DJ77, Serine palmitoyltransferase, sphingolipids, gene

Description

A gene coding serine palmitoyltransferase of Sphingomonas Chungbukensis, a gene encoding serine palmitoyltransferase, a recombinant vector thereof, a transformant, a serine palmitoyltransferase protein using the same and a method for preparing the same DJ77, recombination vector, transformation organism using thereby, serine palmitoyltransferase protein and the preparation method

1 is a view showing a process of manufacturing a shotgun library (Shotgun library),

2 is a diagram illustrating a process of manufacturing a fosmid library,

3 is a diagram summarizing the process of predicting serine palmitoyltransferase (SPT) gene,

4 is a diagram comparing the homology of serine palmitoyltransferase (SPT),

5 is a diagram showing the results of performing 12.5% SDS-PAGE to confirm the serine palmitoyltransferase (SPT) protein,

6 is a diagram showing the results of measuring the activity of serine palmitoyltransferase (SPT) protein using a liquid scintillation spectrometer counter.

The present invention relates to a gene encoding serine palmitoyltransferase (SPT) in Sphingomonas Chungbukensis DJ77.

After Johann L. Thudichum first discovered sphingolipids in brain tissue in 1884, the sphingolipids, which had been thought only as cell membrane components, have been identified by Hannun and Bell. In 1987, the release of sphingolipids inhibited protein kinase C (PKC), which led to a renewed recognition of sphingolipids as active molecules in vivo that regulate biological functions in components of cell membranes. Recently, studies on sphingolipids, which are representative of apoptosis-inducing substances and apoptosis-signaling agents represented by ceramide and sphingosine, have been actively conducted, and sphingosine 1-phosphate. Attention has been focused on the function of (S 1P)) as an intercellular signaling agent. In particular, the role of sphingolipids in the physiological activity regulating function as a signal transduction material has recently attracted attention due to the very high industrial use value.

Although sphingolipids are known as essential components of cell membranes in eukaryotic cells, they are rarely found in bacteria. However Sphingomonas (Sphingomonas) species, while Gram-negative bacteria two kinds generally configure the extracellular polysaccharide (lipopolysaccharide) instead ABONDANCE ping lipid (glycosphingolipid) layer having a cell membrane constituent cell membrane component has a seuping lipid biosynthetic pathway (Ingar, O. et al., Anaerobe, 07 , pp 103-112. 2001). However, until now, only studies on the sphingolipids of bacteria have been conducted on the structure of glycosphingolipids, and the biosynthesis related genes of the sphingomonas paucimobilis ( Sphingomonas paucimobilis) The serine palmitoyltransferase 1 (SPT1) gene is unique (Hiroko I et al., J. Biol. Chem., 276, pp18249-18256. 2001). This may be because the glycosphingolipid biosynthesis pathway of the bacteria is not yet known, and thus, a conventional gene comparison method cannot easily find a related gene, or a gene related to biosynthesis is a new gene that has not yet been identified. In the same eukaryotes, mammalian and yeast, sphingolipid biosynthesis pathways are very different, so in mammals, sphinganine and dihydroceramide are respectively called sphinginine 1-phosphate ( Whereas sphinganine 1phosphate and ceramide take the route, yeast goes through the paths where sphinginine and dihydroceramide are made, such as phytosphingosine and pytoceramide. Therefore, the sphingolipid biosynthesis related genes and pathways of bacteria are also expected to be significantly different from previously known eukaryotic cells. This increases the possibility of producing new forms of sphingolipids or their by-products.

In particular, the sphingoid base, known as the basic unit of sphingolipids, includes sphingosine, dihydrosphingosine and phytosphingosine, which are combined with fatty acids and amides of different structures. It is known to produce various kinds of ceramides. The ceramides thus produced are known to form sugar spingolipids having a more complicated structure by a series of chain reactions with sugar chains, or to combine with phosphocholine to produce sphingomyelin. These substances are known for their excellent skin regeneration, antimicrobial, anti-inflammatory, skin protection and retention effects. By using these properties, by-products and final products produced during the sphingolipid biosynthesis process are widely used as main ingredients such as atopic skin treatments, skin moisturizers, and skin inflammation treatments.

However, the production of such materials currently relies on chemical biosynthesis, making it difficult to produce only the desired materials, which requires a lot of time and cost in the post-production purification process. Therefore, there is an urgent need for a new method of producing sphingoid bases with high industrial utility.

Sphingomonas A new form of sphingolipids can be found by discovering the sphingolipid biosynthesis pathway of bacteria through the production of sphingolipids for mass production of sphingolipids, which overexpress SPT, a key enzyme in the sphingolipid biosynthesis step in the genus bacteria. The manipulation of sphingolipid biosynthesis regulatory genes may lead to the accumulation of techniques that can increase the desired product or simplify the recovery of the product.

Serine palmitoyltransferase is an enzyme that turns L-serine and palmitoyl-CoA into 3-ketodihydrosphingosine (KDS) and is known to be involved in the first stage of sphingolipid biosynthesis. . In bacteria, since it was first discovered in S. paucimobilis by Hiroko in 2001, it has not been found in other bacteria.

Therefore, the present inventors were first isolated by the present inventors using SPT information of S. paucimobilis ( S. paucimobilis) sphingomonas Chungbuk reported as a new species in the International Journal of Systematic and Evolutionary Microbiology (IJSEM) The present invention was completed by finding a serine palmitoyltransferase gene in Kensis DJ77 and overexpressing it.

An object of the present invention is to identify the sphingolipid biosynthesis pathway of bacteria and provide a desired sphingolipid metabolite by providing a serine palmitoyltransferase gene sequence, which is a key enzyme of the sphingolipid biosynthesis step in Sphingomonas Chungbukkensis DJ77. Biochemically enabling synthesis to broaden the industrial use of sphingolipids.

In order to accomplish the above object, the present invention provides a serine palmitoyltransferase (SPT) gene sequence of Sphingomonas Chungbukkensis DJ77 described in SEQ ID NO: 1.

The present invention also provides a gene sequence encoding serine palmitoyltransferase (SPT) protein having at least 90% homology with the gene sequence set forth in SEQ ID NO: 1.

The present invention also provides an expression vector containing the SPT gene sequence of Sphingomonas Chungbuk Ensis DJ77 and a method for producing the same.

The present invention also provides E. coli transformed with an expression vector containing the SPT gene sequence of Sphingmonas Chungbukkensis DJ77.

The present invention also provides a serine palmitoyltransferase (SPT) protein comprising the amino acid sequence of SEQ ID NO: 2 and a method for preparing the same.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The sphingomonas chungbukensis DJ77 strain used in the present invention is an aerobic gram-negative bacterium isolated from the sewage and wastewater of Daejeon Industrial Complex in 1986 by IJSEM (2000). A new species has been reported in the International Journal of Systematic and Evolutionary Microbiology (Kim et al., 2000). The strain is known to have the ability to degrade phenanthrene, biphenyl, salicylate, p-hydroxybenzoate, and benzoate, which are hardly degradable aromatic hydrocarbons. (Kim et al , 1997), biopolymer synthesis such as PHB (polyhydroxybutyrate), EPS (exopolysaccharide), etc., and is known to have the ability to biosynthesize sphingolipids, L-serine and palmitoyl ( It has been shown to produce the serine palmitoyltransferase (SPT) enzyme, which makes palmitoyl) -CoA into 3-ketodihydrosphingosine (KDS).

The enzyme is known to be involved in the first stage of sphingolipid biosynthesis. This is not only a gene demonstrating that this strain produces sphingolipids unlike other Gram-negative bacteria, but also explains that the DJ77 strain has a sphingolipid biosynthesis related gene in addition to the SPT gene. It is expected that the sphingolipid biosynthesis process can be predicted through the SPT gene manipulation, and the study on the regulation of the sphingolipid lipid production through the SPT gene engineering increases the industrial applicability of the gene of the present invention.

Meanwhile, the gene encoding the SPT uses ATG as an initiation codon, TGA as a termination codon, and has a ribosomal binding site called GGA in front of the start codon 6 bp and a total of 1218 bp of open reading prime. frame).

In addition, the present invention provides an expression vector containing the SPT gene sequence of sphingomonas Chungbukkensis DJ77, a method for preparing the same, and E. coli transformed using the same.

The method for producing the transformed Escherichia coli is as follows.

SPT gene is amplified by using a PCR method commonly used as a primer prepared by adding restriction enzymes. At this time, the denaturation process is about 80-100 ℃, preferably at a temperature of 94 ℃, for about 10-60 seconds, preferably 30 seconds, the annealing process is about 50-80 ℃, Preferably at a temperature of 67.5 ° C. for about 10-60 seconds, preferably 30 seconds, the extension process is carried out for 1 minute-10 minutes, preferably for 1 minute and the process is carried out 10-50 times, Preferably repeat 30 times. After the amplified gene and the vector are cut with the same restriction enzyme, ligation is performed to prepare an expression vector, which is inserted into E. coli by electroporation to transform E. coli.

The vector and E. coli can be used for all commonly used vectors and E. coli, preferably, the vector uses a pET series (series), pTA1529, pBAce, pGEX series, pMAL series, pTrx, pTrxFus and the like E. coli is BL21 (DE3) pLysS, JM101, DH5F ', TG1, etc. are used.

Escherichia coli BL21 (DE3) pLysS transformed with the expression vector pET21c containing the SPT gene sequence of the present invention was deposited on February 03, 2005 to KCTC (Accession No. 10777BP). It will be apparent to those skilled in the art that the SPT gene of the present invention can be easily obtained by culturing the deposited Escherichia coli through a conventional DNA preparation process.

The present invention also provides a serine palmitoyltransferase (SPT) protein comprising the amino acid sequence of SEQ ID NO: 2 coding with the gene of SEQ ID NO: 1 and a method for preparing the same.

The SPT protein is obtained through conventional culture and obtaining methods in the art using E. coli transformed with an expression vector containing the SPT gene sequence of Sphingomonas Chungbukkensis DJ77.

After culturing the transformed Escherichia coli in a basic nutrient medium, preferably LB medium, to which an antibiotic, preferably streptomycin, tetracycline, ampicillin, chloramphenicol, X-gal and IPTG is added, centrifugation The whole cellular protein (whole cellular extract) can be obtained, and the SPT protein can be isolated and obtained using protein separation methods such as SDS-PAGE, isoelectric focusing (IEF), and peptide mass fingerprints.

The present invention will be described in more detail based on the following examples and experimental examples, but the present invention is not limited thereto.

Reference Example 1. Strains and Vectors

The strain used in this experiment was Sphingomonas chungbukensis DJ77 (KCTC 2955), isolated and reported from sewage and wastewater from Daejeon Industrial Complex, and was used to produce 1-2 kb library of DJ77. The vector was pBluescript II SK (-) (Stratagene Co., USA) and the vector used to construct the 3-4 kb library was pUC19 (Takara, Japan). Their host strain is E. coli XL1-Blue (Stratagene Co., USA) was used and E. coli EPI300 ^™ -T1 ^R (Epicentre, USA) and pCC1FOS ^™ (Epicentre, USA) were used as phosphide library construction strains and vectors. E. coli XL1-Blue (Stratagene Co., USA) was used as a host strain for subclone production of the Fosmid clone and pBluescript II SK (-) (Stratagene Co., USA) was used as a vector. E. coli BL21 (DE3) pLysS (Novagen, Germany) was used as the expression host strain of the target protein (Serine palmitoyl transferase), and pET21c (Novagen, Germany) was used as the expression vector.

Reference Example 2. Reagents

Among the media components used in this experiment, tryptone and yeast extract were from Difco Laboratories (Detroit, Mich, USA), and agar powder was from Sangjun Co., Ltd. (Kyongkide, Poungtack, NaCl is Shingo pure chemicals Co., Ltd. It was purchased from (Osaka, Japan). Restriction enzymes required for DNA manipulation were purchased from Roche (Mannheim, Germany) and Takara (Otsu, shiga, Japan). Reagents such as Tris, EDTA and sodium dodecyl sulfate (SDS) used in the rest of the experiment were purchased from Sigma (St. Louis, Mo, USA).

Reference Example 3. Use Badge

The basic nutrient medium is Luria-Bertani (LB), which contains 1% (w / v) tryptone, 0.5% (w / v) yeast extract and 0.5% NaCl in 1 liter of distilled water. Solid medium was used to add agar powder to 1.5% (w / v). The medium used in each experiment was selected and used according to the purpose of the experiment and the characteristics of the strain, and the final concentration of β antibiotic and reagent added to the medium is as follows. Streptomycin (50 mg / l), tetracycline (15 mg / l), ampicillin (100 mg / l), chloramphenicol (12.5 mg / l), X-gal (5-bromo-4-chloro-3-indolyl- β- D-galactoside; 50 mg / l) and IPTG (isopropyl- β- D-thiogalactoside; 50 mg / l) were added to the medium according to the experimental purpose.

Experimental Example 1. Prediction and Acquisition of Serine Palmitoyltransferase (SPT) Gene

In order to analyze the genome sequence of Sphingmonas Chungbuk Kensis DJ77, a Shotgun library and a Fosmid library were prepared (see FIGS. 1 and 2). The shotgun library was produced in two sizes of 1-2 kb and 3-4 kb using a sonicator, and the phosmid library was produced using a kit (CopyControlTM Fosmid Library Production Kit, Epicentre, USA). . The clones obtained through library production were commissioned by Takara to obtain 19,576 sequence files, and then the genes were predicted using various bioinformatics tools. did. First, the quality of the sequence was checked using PHRED (obtained: University of Washington website), and the vector sequence was removed using CROSS_MATCH (obtained: University of Washington website). The pure sequences of sphingomonas Chungbuk Nsis DJ77 thus obtained were combined and edited in contig (multi fasta file format) using PHRAP (obtained from the University of Washington website). The produced contig searched ORFs using ORF finder (http://ncbi.nlm.nih.gov) and Glimmer (http://www.tigr.org). ORFs performed functional prediction via BLASTP (http://ncbi.nlm.nih.gov) (see FIG. 3). Through this, the SPT gene, which is related to sphingolipid biosynthesis, was obtained, and the FA153 clone including the entire SPT gene was found. The FA153 clone was digested with various restriction enzymes such as Bam H I, Hin d III, Eco R I, and a subclone was cloned and sequenced by Takarasa. Through this, the entire SPT gene of Sphingomonas Chungbuk Kensis DJ77 was obtained.

Experimental Example 2 Homology Comparison of Serine Palmitoyltransferase (SPT)

Similarity test results of the amino acid sequences constituting serine palmitoyltransferase (SPT) of sphingomonas Chungbukkensis DJ77 and serine palmitoyltransferase 1 of sphingomonas porchmobilis EY2395T using BLASTP The two genes showed 75% similarity (see Table 1). In addition, the active site was predicted through the gene homology test program Clustalx (obtained: http://www.igbmc.u-strasbg.fr/Bioinfo/). This allows PLP binding, the active site of the AONS (8-amino-7-oxononanoate synthase) protein of E. coli , an enzyme that catalyzes the condensation reaction of _L -alanine and pimeloyl-CoA. It was confirmed that the lysine of the binding site was well preserved (see FIG. 4).

Source Locustag / gene name Product Identities (%) Positives (%) Gene Bank accession No. Z. mobilis subsp . mobilis ZM4 ZMO1270 Serine palmitoyltransferase 78 88 YP_163005 S. paucimobilis SPT1 Serine palmitoyltransferase 75 86 BAB56013 N. aromaticivorans DSM 12444 Saro02003827 COG0156: 7-keto-8-aminopelargonate synthetase and related enzymes 74 84 ZP_00301852 C. crescentus CB15 CC1162 Aminotransferase, class II 52 72 NP_419978 A. vinelandii Avin02002486 COG0156: 7-keto-8-aminopelargonate synthetase and related enzymes 48 66 ZP_00090424 N. europaea ATCC 19718 NE1655 Aminotransferases class-I 45 64 NP_841689 Escherichia coli ORF101 hypothetical transferase Hypothetical protein 46 64 CAE85251

Experimental Example 3 Preparation of Recombinant Vector and Transformant

In order to clone the serine palmitoyltransferase (SPT) gene into the pET21c expression vector, a primer was prepared by adding restriction enzyme Bam H I / Hin d III.

The prepared primer is as follows.

SPTEX21c L: 5 '-ACT GGA TCC ATA TGA CCA GCA ATA ACC GC-3',

SPTEX21c R: 5'- TAT AAG CTT GCA GAT CGC GCC TGT CGC C-3 '.

PCR was performed by a method commonly known as the primer prepared as described above. The denature process was repeated for 30 seconds at 94 ° C, the annealing process for 30 seconds at 67.5 ° C, and the extension process was repeated 30 times for 1 minute, and the last extension process was performed for 10 minutes. Each gene was amplified by PCR, cut with restriction enzyme Bam H I / Hin d III, and the DNA was purified and ligation was performed on the expression vector pET21c (Novagen, Germany). Electroporation is carried out using a 1 mm gap cuvette under conditions of voltage 1.8 kV, capacity 25 mA and resistance 200 ohms to E. coli BL21 (DE3) pLysS. By transformation. At this time, the electroporation machine used ECM 630 of BTX (USA).

Experimental Example 4. Measurement of protein expression and activity

4-1. Protein Expression and Isolation Purification

In order to confirm the function of the cloned gene, the target protein was expressed and purified.

Serine palmitoyltransferase (SPT) protein was induced at 37 ° C by adding 1 mM IPTG (Sigma, USA) in E. coli BL21 (DE3) pLysS, with 6 · His + attached to the N-terminal. Mass expression. E. coli cells induced protein expression was pulverized ultrasonically, and purified using a metal chelated resin (ProBondTM Nickel-Chelating Resin). In order to observe the induction and efficiency of IPTG and the purity of the purified protein, 12.5% SDS-PAGE was performed to confirm the pure SPT protein at 37 kD (see FIG. 5).

4-2. Serine Palmitoyltransferase (SPT) Assays

SPT assay was performed to measure the activity of the protein expressed in Experimental Example 4-1.

First in assay buffer (100 mM HEPES pH 8.3, 5 mM DTT, 2.5 mM EDTA pH 7.0, 50 μM Pyridoxal phosphate, 200 μM Palmitoyl-CoA, 1 mM labeled L-serine (L- serine) [ ³ H]) and 100 μg of SPT protein sample were mixed to 100 μl and reacted at 37 ° C. for 15 minutes. To this was added 1.5 ml of chloroform: methanol (1: 2, v / v) and vortexed, centrifuged for 1 minute, followed by 1 ml chloroform and 2 ml of 0.5 N NH ₄ OH. It was scanned. It was centrifuged for 5 minutes, the supernatant was removed and placed in 2 ml D. W for washing, then vortexed and centrifuged for another 5 minutes. 800 μl of the lower layer of chloroform was dried, resuspended in 4 ml of scintillation fluid (Perkin elmer, USA), and then liquid scintillation spectrometer counter (Model: wallac 1408 DSA, manufacturer: PerkinElmer). (USA)) to measure activity.

Sphingomonas Chungbuk Ensis DJ77's total protein, sphingomonas pochimobilis ATCC 31461 all protein known as similar strain, whole protein of SPT overexpressing E. coli strain, purified SPT protein, overexpressing E. coli without SPT protein The SPT activity of the whole protein of the strain was measured by a liquid scintillation spectrometer counter. As a result, the protein of the SPT overexpressing strain showed about 100 times more activity than that of other strains not overexpressing. Considering the difference in the total protein amount showed more than 10,000 times increased activity. This confirms that the protein is actually SPT which condenses L-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine (see FIG. 6).

Gene encoding SPT in sphingomonas Chungbukkensis DJ77 of the present invention enables expression of serine palmitoyltransferase, a key enzyme of sphingolipid biosynthesis, through recombinant vectors and transformants. The discovery of high lipid biosynthetic pathways and the manipulation of sphingolipid biosynthesis regulatory genes are expected to allow for the accumulation of technologies that can increase the desired product or simplify the recovery of the product. In particular, sphingolipids have been recognized for their high business value in the world, but their types and characteristics are not well known, so research on new forms of sphingolipids in bacteria is of great value. The genes of the invention will be of great use in this study.

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Claims (8)

The serine palmitoyltransferase (SPT) gene of Sphingomonas chungbukensis DJ77 of SEQ ID NO: 1. delete An expression vector containing the gene of claim 1. A method for producing an expression vector comprising the gene of claim 1. E. coli transformed with the expression vector containing the gene of claim 1. Serine palmitoyltransferase (SPT) protein comprising the amino acid sequence of SEQ ID NO: 2. delete A method for producing a serine palmitoyltransferase (SPT) protein, comprising culturing Escherichia coli according to claim 5.

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