KR100196768B1 - Sec y gene which secrets protein in streptomyces and mass production method - Google Patents

Abstract

S. coelicolor, which is a gram-positive organism such as B. subtilis and M. luteus, has been developed as a natural system of protein secretion, produces a variety of useful secondary metabolites and is used as a molecular system for actinomycetes. The SecY protein gene, which is known to act as a trans locator for extracellular protein secretion from Streptomyces lividans, which is widely used as an expression strain, is isolated and compared with the secY gene sequence so far found to be homologous, homologous and hydrophobic (SecY) gene by hydrophobic analysis, and to develop S. coelicolor and S. lividans, which have developed a natural protein secretion system by using this secY gene, as a strain to increase useful heterologous protein secretion.

Description

SecY gene involved in the secretion of actinomycetes and a method for mass production of proteins using the same

Figure 1 shows a PCR primer made using the amino acid sequence of the region conserved in the spc operon.

2 shows a PCR product obtained from the genomic DNA of Streptomyces coelicolor (a) and Streptomyces lividans (b) using four primers.

Figure 3 shows the restriction map of secY gene cloned in Streptomyces coelicolor (a) and Streptomyces lividans (b).

FIGS. 4 (a) and 4 (b) are diagrams showing DNA sequences and deduced amino acid sequences of Streptomyces coelicolor and Streptomyces lividans secY genes. FIG.

FIGS. 5 (a) and (b) show Streptomyces coelicolor, Micrococcus luteus, and Bacillus subtilils.

6 (a) and 6 (b) are diagrams showing the amino acid sequence of the SecY protein in Streptomyces lividans.

(A), (b), (c), and (c), respectively. (A) shows M. luteus and B. subtilis, (b) shows M. luteus and S. coelicolor, Lt; RTI ID = 0.0 > SecY < / RTI >

FIG. 8 is a comparison of the hydrophobicity of SecY proteins in B. subtilils (a), M. luteus (b), S. coelicolor (c) S. lividans (d)

FIG. 9 is a diagram showing the degree of amino acid similarity between the cytoplasmic region of the SecY protein, which is well-preserved in Streptomyces coelicolor and Micrococcus luteus,

[Industrial Applications]

The present invention relates to the mass secretion of proteins, and more particularly, to the isolation of genes that play an important role in the secretion of cellular proteins in actinomycetes such as Streptomyces coelicolor and Streptomyces lividans and the use of this gene for the secretion of useful proteins in actinomycetes The possibility of the development of the strain.

BACKGROUND ART [0002]

Streptomyces belonging to the group of gram-positive sedentary bacteria is well known as a strain producing various secondary metabolites which are easy to separate from soil and industrially useful. This Streptomyces grows in the form of substrate mycelium according to the nutritional environment, and when the nutrients are depleted, it forms aerial mycelium and segregates into several spores by forming septa in the mycelium. According to recent molecular biology research, the cell differentiation of Streptomyces becomes more active in the stasis where the cell differentiation reaction occurs in the cell growth period, and the production of the secondary metabolite used as a useful substance such as antibiotics is mainly biosynthesized in the stasis It is known that when mutations occur in the genes expressed at these stages, antibiotic production ability and spore-forming ability are frequently lost, and secondary metabolism and morphological differentiation in Streptomyces are closely related to each other. At present, studies on the regulation of cell differentiation and production of secondary metabolites by environmental factors are simultaneously being conducted.

Recently, the knowledge about the regulation of the fermentation process for the production of antibiotics has been accumulated. Therefore, it is attracting great interest to use Streptomyces, which produces about 60% of the antibiotics currently used, as a host cell for the expression of xenogeneic cell protein.

However, such actinomycetes secrete many cell enzymes other than secondary metabolites. However, there is no study on the mechanism of this protein secretion yet, and in order to increase useful protein production, overexpression of the substrate involved in the synthesis pathway, But it still has a lot of problems. However, it would be very useful in industry to develop mass-producing strains of proteins using the seeY gene, which plays an important role in protein secretion, and this has already become possible in experiments using the secY gene of Bacillus subtilis.

In the meantime, studies on the protein secretion mechanism of prokaryotic cells related to the secretion of protein have been carried out on Escherichia coli, which is gram-negative bacterium, to some extent, and secA, secB, secD, secE. However, there is almost no research on the mechanism of protein secretion in Gram-positive bacteria. Among them, the isolation of secY gene as a protein transcriptor in Bacillus subtilis performed by Sub et al. (1990) (Sec.), SecY gene, and the isolation of secY gene from several strains such as Micrococcus luteus.

SecY expressed by the secY gene is a membrane protein that plays an important role in protein secretion and cell growth. In the case of Escherichia coli mutated in the secY gene, secretory protein can not be secreted outside the cell, and the precursor of the protein is accumulated in the cell . This secY has a topology in which the NH ₂ - and COOH- ends are directed to the cytoplasm in the cell membrane, and a portion thereof passes through the cell membrane 10 times.

[Object of the invention]

DISCLOSURE OF THE INVENTION An object of the present invention is to solve the above-mentioned problems of the prior art, and it is an object of the present invention to provide a stactobacterium which is used as a host strain for producing many kinds of useful heterologous proteins, a secY gene capable of identifying a protein secretion mechanism not yet revealed in Streptomyces And to develop actinomycetes as protein secretion strains by using this gene to increase useful protein production.

SUMMARY OF THE INVENTION [

In order to solve the above-mentioned object of the present invention, the present invention has the following configuration.

In the present invention, a secY gene serving as a transpos locus important for protein secretion is provided.

The secY gene is preferably isolated from Streptomyces coelicolor and Streptomyces lividans. The secY gene has the nucleotide sequence of Figure 4 and is included in the adk operon. Also, the promoter region of secY gene is presumed to be a region having AGGA at -10 position from the GTG translation initiation codon and TTGACG nucleotide sequence at -35 position, and the ribosome binding region is also a region having GGAGG nucleotide sequence from ORF to -10 position It is 1314 bp from the GTG start codon to the TGA termination codon and consists of 438 amino acids and has a molecular weight of 47.4 kD. It is also a transmembrane protein with a structure in which ten hydrophobic transmembrane segments pass through the cell wall ten times.

In the present invention, the secY gene is isolated and the plasmid pGEMC-secY and pGEML-secY vectors containing the DNA fragment in which the gene sequence is determined are provided in E. coli.

The present invention also relates to a method for mass secretion of a heterologous protein produced by inserting a secY gene into E. coli and actinomycete tufty vector DNA, introducing a vector DNA into which a secY gene has been inserted into a host cell, culturing the host cell, pKCY11. The present invention also includes a vector or strain using a modification of the secY gene to increase protein secretion. The host cell is preferably actinomycetes.

The secY protein has the amino acid sequence of FIG. 5.

The following examples are provided to further illustrate the present invention. However, the present invention is not limited to the following examples.

[Example]

I. Culture of Streptomyces coelicolor

Solid medium in R ₂ YE medium (sucrose (sucrose) 103g, potassium sulfate (K ₂ SO ₄₎ 0.25G, magnesium chloride 6 beard _{(MgCL 2 · 6H 2 O)} 10.12g, glucose (glucose) 10g, Casa Mino Acid 5 g of yeast extract, 5 g of TES, 0.5% of trace elements, 1 ml of potassium primary phosphate (KH ₂ PO ₄ ), 2 ml of 1 M calcium chloride dihydrate, 1.5 ml of 20% L- Sodium hydroxide 0.7 ml per 100 ml Streptomyces coelicolor (Muller: hereinafter referred to as S. coelicolor) was cultured in 2 ml agar 25 g per liter.

II. DNA Isolation and Recombination

The isolation of chromosomal DNA from S. coelicolor was performed by modifying the method of Hopwood et al. (23).

YEME liquid medium (3 g of yeast extract, 5 g of peptone, 2 g of malt extract, 10 g of glucose, 340 g of sucrose, 50 ml of 2.5 M magnesium chloride hexahydrate (2 ml / per liter after high pressure autoclave) Lt; 0 > C for 2 to 3 days.

Thereafter, the cells were centrifuged at 8,000 rpm to discard the confluent solution. The cells were washed once with lysis buffer (lysis buffer: 15% sucrose, 25 mM Tris-Cl pH 7.5, 25 mM EDTA) and centrifuged again at 8,000 rpm . The cells were suspended in 20 ml of Lysozyme buffer containing 5 mg / ml of Lysozyme and reacted at 37 ° C for 1 hour. Thereafter, 1 ml of 10% SDS was added and the mixture was allowed to stand at 70 ° C for 30 minutes. Then, an equal amount of phenol / / chloroform) and centrifuged at 12,000 rpm. Transfer the supernatant to a new tube, add 2x ethanol and 0.1x 5 M potassium acetate, spool out the DNA with a glass rod, ). The DNA obtained by the above procedure was washed with 70% ethanol, dried and dissolved in TE buffer (10 mM Tris-Cl pH 7.5, 1 mM EDTA).

Plasmid isolation, DNA recombination and transformation in E. coli were carried out according to the method of Maniatis et al. (24).

III. Polymerase Chain Reaction (PCR)

1. Preparation of PCR primer

The amino acid sequence of the L15 gene in the spectinomycin (spc) operon of E. coli, B. subtilis, and M. luteus was compared with the amino acid sequence of the conserved region in the secK region and the adk region of the adk operon, The nucleotide sequence of the PCR primers was determined by considering the sequence and considering the GC ratio (70-74%) of Streptomyces and the frequency of codon usage.

The prepared PCR primer

A synthesizer of Pharmacia was prepared using DNA.

FIG. 1 shows the amino acid sequences of the ribosomal proteins L15, secY and Adk of Micrococcus luteus (ML), Bacillus subtilis (BS) and Escherichia coli (EC). The area inside the box shows the amino acid in the primer synthesis region.

Comparing the amino acid sequence of the SecY protein, the correlation between Gram-negative bacilli and gram-positive B. subtilis was highly preserved, and among the ten membrane-spanning segments, Region, and it was found that this region plays an essential role in protein secretion mechanism among secY proteins. In order to isolate such secY gene from actinomycetes, two primers sp1 and sp2 were prepared in the 2nd and 5th segments, where SecY protein of various strains has excellent conservation of amino acid sequence. In addition, sp3, which has the same sequence as sp2 in the opposite direction, was also prepared.

In addition, among the secY genes isolated so far, most secY genes including the E. coli secY gene are present in the spc operon and exist between the L15 and adk genes, and the gene organization around them is also highly similar. Two primers were constructed in the region of ribosomal protein L15 and adenylate kinase (a) at both sides of the gene and PCR experiments were performed.

2. PCR reaction

50 ng of template, 10 x Taq DNA polymerase, 4 μl of 25 mM dNTP, 50 pmol of each primer, 25 mM chlorine (50 mM) of each primer was added to a total volume of 50 μl according to the method of Atlas (RM) and Bej The mixture of 1 μM of magnesium and 1 μM of the DNA polymerizing agent was denatured at 94 ° C. for 1 minute and 30 seconds using a PCR machine of Perkin Elmer Cetus and annealed at 55 ° C. for 30 seconds. Followed by extension at 72 ° C for 2 minutes and 20 seconds. This procedure was repeated 35 times, followed by extension reaction at 72 ° C for 15 minutes, and PCR products were confirmed using 1% agarose gel. The results are shown in FIG. Lane 1 is a size marker, λ-DNA digested with BstEII, lane 2 is a PCR product using L15 and Adk primer, lane 3 is a PCR product using L15 and sp2 primers, sp3 and Adk primers.

PCR was performed by combining four PCR primers (L15, SP1, SP2, Adk primer) prepared for cloning secY gene from the chromosomal genomic DNA isolated from S. coelicolor, PCR products were obtained. The two amplification fragments obtained in this experiment were designed as amplified fragments of about 1.9 kb, the expected size when using the L15 and Adk primers, and about 1.1 kb, the expected size when using the L15 and SP2 primers, lost. In S. lividans, an amplified fragment of about 1.9 kb size, which was expected to be the L15 and AdK primer, and an amplified fragment of about 800 bp, was obtained from the sp3 and Adk primers. The amplified fragment was expected to contain the secY gene to be cloned and subcloned into a pGEM-T vector for sequencing.

3. Sequence analysis of secY gene

The restriction fragments of PstI, SmaI, PvuII, KpnI and SacI were used to determine the nucleotide sequence of the PCR product. Small fragments were obtained by restriction enzyme mapping to obtain pBluescriptII SK (+) or M13mp18 or mp19 vector Cloning was performed and the nucleotide sequence was determined using an automatic sequencer from Pharmacia, which is shown in FIG.

Sequence analysis was performed using DNA / protein sequence analysis software DNASIS / PROSIS (Hitachi Software Engineering) and GeneBank. The determined nucleotide sequence was analyzed by DNASIS and the region encoding the protein was searched to confirm the presence of one complete ORF. This ORF has a TTGACG nucleotide sequence at position-AGGGA and -35 at the -10 position from the GTG translation start codon, similar to GAGGAT / TTGACG, the nucleotide sequence of the ermE promoter region of the resistance gene of the erythromycin producing strain, Sacharopolyspora erythraea. This secY gene is considered to be a promoter region and a ribosomal binding site (GGAGG) is located near the -10 region from ORF (6). The secY gene was composed of 1314 bp base and 438 amino acids from the GTG start codon to the TGA end codon, and the GC content was more than 70%, showing the typical appearance of actinomycetes (FIG. 4). The ribosome binding sites deduced in FIG. 4 are underlined.

Analysis using DNASIS showed 58% similarity in nucleotide sequence comparison with M. luteus with similar GC content and 40% similarity with B. subtilis with 45% GC content, while amino acid In comparison of sequence, 39.7% showed high conservation, and the use of biased codon of actinomycetes was confirmed. Actinomycetes are synthesized by incorporating G or C of about 50% in the second base, which plays a crucial role in amino acid designation, in the third base of the codon, which has an average GC of more than 70% It is reducing the biased amino acid content of intracellular proteins. Therefore, the conservation of the nucleotide sequence between B. subtilis and S. coelicolor or S. lividans secY was about 40%.

However, amino acid sequence showed a relatively high homology of 38.7% with B. subtilis and 56% with M. liteus. On the other hand, the nucleotide sequences of S. coelicolor or S. lividans were completely identical in the secY gene, but one or two bases were different in the C terminal region of the L15 gene, which is the upper part of the secY gene. This result can be seen from FIGS. 5 and 6. In Fig. 5, the alignment in parentheses indicates 10 cell membrane spanning segments. In Fig. 6 (a), M. luteus and B. subtilis, (b) M. luteus and S. coelicolor, and (c) M. luteus and S. lividans, (d) are graphs showing the similarity between the amino acid sequences of S. coelicolor and S. lividans.

4. Hydrophobicity analysis of S. coelicolor secY

Both H. subtilis and M. luteus SecY proteins, which are gram-negative bacteria and Gram-positive bacteria, are directed to the cytoplasm of NH ₂ - and COOH- ends, pass through the cell membrane 10 times, It is known that six domains are the membrane proteins, which are the domain and the part facing the cell interior.

In order to confirm that the gene isolated in the above process is secY gene, the hydrophobicity was analyzed using the amino acid sequence in the Kyte-Doolittle plots of the PROSIS program. FIG. 7 shows a very similar hydrophobic profile of B. subtilis, M. luteus, S. coelicolor and S. lividans SecY proteins, and showing Kitty-Durtle curve using 14 windows. Each contains 10 potential membrane-spanning segments. As a result of amino acid sequence analysis, the molecular weight of S. coelicolor and S. lividans SecY protein was 47.4 kD, and as shown in Fig. 7, there were 10 hydrophobic regions with similar pattern to gram-positive bacteria B. subtilis and M. luteus. . The SecY protein of coelicolor and S. lividans was also found to be a transmembrane protein that passed through the cell wall 10 times. In the luteus, the 2nd, 5th and 10th regions among 10 cell membrane spanning segments were particularly well preserved (Fig. 8). It is thought that this region plays an important role in protein secretion. In Fig. 8, the cytoplasmic regions and cell membrane-spanning segments of S. coelicolor and M. luteus SecY proteins are very well preserved, and the secY with ten transmembrane segments marked with a hatched box and the outer Region and the end regions of NH ₂ - and COOH-. The value of each region is the percentage of amino acids divided by the corresponding region from S. coelicolor.

As a result of the comparison of the genetic organization of E. coli, B. subtilis, M. luteus and S. coelicolor secY gene, the L15-secY-α operon (a ribosomal protein containing an RNA polymerase α-subunit Operon) and gram-positive bacteria B. subtilis, M. luteus, S. coelicolor and S. lividans were composed of L15-secY-adk operon-α operon. Among the Gram-positive bacteria with the same gene structure, the B. subtilis actually contained 30 bp of the ribosomal proteins present in the spc operon, but in the case of L15 and secY, the gap was 1 bp and the adk gene was 75 bp And secY of B. subtilis was found to be present in the spc operon similar to E. coli as a whole, although in the previous experiment, a minor promoter expressed in the exponential period of cell growth period was found before the secY gene. However, Ohama (T) et al. Have included their promoters in front of secY at intervals of L15 and 162 bp for secL of M. luteus and 52 bp for the adk gene, and the appropriate ribosome binding site It is not present and rather reported to belong to the adk operon. S. coelicolor and S. lividans, which have the same GC content and similar GC content, have a large interval of 257 bp from L15, unlike M. luteus, and the adk gene and the secY codon termination codon have one initiation codon and one base of adk gene S. coelicolor secY belongs to the adk operon rather than the spc operon more clearly because it has a shared and internally coupled structure.

This analysis shows that the secY gene, which is a gram-negative bacterium, exists in the spc operon, and the secY subregion is located in the spc operon when the α-operon is located not in the adk operon but in B. subtilis in gram-positive bacteria However, it has its own minor promoter and the adk gene is located in the subregion. However, in M. luteus, it is similar to S. coelicolor and S. lividans in the order of L15-secY-adk, but in the order of B. subtilis, the secY gene has its own promoter region and is separated from the spc operon but rather close to the adk operon And S. lividans in the case of S. coelicolor and S. lividans share a single base with adk gene and have a structure that is coupled to the entire plant. As a result, secY gene is not contained in the spc operon but rather in the adk operon Can be seen as including.

(Function of the Invention)

The separation of secY gene from actinomycetes, which foreign companies are attracting attention as a heterologous protein expression and secretory host strain, can provide an important starting point for studying the mechanism of protein secretion and regulation of useful protein secretion of actinomycetes, The surrounding genetic structure is essential components of the ribosome. Protein secretion and energy storage factor are essential constituents of the cell such as ATP-forming protein. It is also important for classification of bacteria that have been morphologically and physiologically changed for a long time. It can be said that it contains a le.

Claims (12)

SecY gene of Streptomyces lividans having the DNA sequence of FIG. 4 (b) involved in the secretion of the protein. The method according to claim 1, wherein the secY gene is a secY gene contained in an adk operon. [Claim 2] The method according to claim 1, wherein the promoter region of the secY gene is a region having AGGGA at position -10 from the GTG translation initiation codon and a TTGACG nucleotide sequence at position -35, and the ribosome binding region has a GGAGG nucleotide sequence at position -10 from the ORF SecY gene which is 1314 bp from the GTG start codon to the TGA termination codon and constitutes 438 amino acids. The secY gene according to claim 1, wherein the secY gene has a molecular weight of 47.4 kD and has ten hydrophobic transmembrane segments. The secY gene encodes a SecY protein, which is a trans membrane protein having a structure that passes through the cell wall 10 times. A plasmid stable in E. coli comprising a DNA fragment obtained by separating the secY gene of claim 1 and determining the gene sequence. A stactobacillus plasmid comprising a DNA fragment obtained by isolating the secY gene of claim 1 and determining the gene sequence. Inserting the secY gene of claim 1 into a vector DNA; Introducing the vector DNA into which the secY gene of claim 1 is inserted into the host cell; A method for mass-producing a protein produced by culturing the host cell. 8. The method according to claim 7, wherein the host cell is a mass secreted protein produced by actinomycetes. A SecY protein which is expressed by the secY gene of claim 1 to secrete a heterologous protein. 10. The method of claim 9, wherein the SecY protein is a SecY protein having the amino acid sequence of FIG. 5. A vector prepared by using and modifying the secY gene of claim 1 for the purpose of increasing protein secretion. A strain produced by using and modifying the secY gene of claim 1 for the purpose of increasing protein secretion.