WO2003106686A1 - Food grade vector comprising psti-spei fragment of pmmh1 plasmid derived from wild-type bacillus - Google Patents

Food grade vector comprising psti-spei fragment of pmmh1 plasmid derived from wild-type bacillus Download PDF

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WO2003106686A1
WO2003106686A1 PCT/KR2003/001170 KR0301170W WO03106686A1 WO 2003106686 A1 WO2003106686 A1 WO 2003106686A1 KR 0301170 W KR0301170 W KR 0301170W WO 03106686 A1 WO03106686 A1 WO 03106686A1
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vector
pmmhl
food grade
plasmid
bacillus
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PCT/KR2003/001170
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French (fr)
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Joo-Won Suh
Young-Yell Yang
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B & C Biopharm
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Priority to AU2003244231A priority Critical patent/AU2003244231A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/15—Vitamins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to food grade vector, and more particularly, to food grade vector comprising fragment of pMMHl plasmid derived from wild-type Bacillus mesentericus.
  • pMMHl is a plasmid isolated from bacteria(5 ⁇ cz ' // «_s' subtilis and its analogues) which has been used for a long time as a major zymogens of the fermentation food made from bean and so whose safety has been recognized internationally and does no harm to human bodies.
  • the present invention constructed various vectors using pMMHl to develop a technique of food grade vector, and intended to find the portion comprising the smallest part of pMMHl altogether with having stability among vectors constructed by this method and then here to construct a expression/secretion vector system producing physiologically useful materials.
  • the object of the present invention is to provide edible food grade vector using plasmid derived from wild-type Bacillus which inhabits in kinds of traditional Korean soy source.
  • the object of the above-mentioned present invention was achieved by isolating the pMMHl plasmid from Bacillus mesentericus KCTC 0750
  • BP to confirm that the plasmid was suitable to use as a food grade vector, constructing a vector capable of being used in both Bacillus and E. coli by deleting each portion of pMMHl one by one and ligating E. coli plasmid, pGEM5z(+) to the portion of pMMHl, transforming Bacillus, a host for the constructed vector, culturing it under condition without antibiotics up to 100 generations in successive batch cultures to obtain stability and confirming that 2.3kb fragment ⁇ tl-S el) was most suitable to be developed as a food grade vector.
  • the present invention composes of two steps; the one is to construct vectors by deleting one by one remnant parts except for replication origin among five portions identified by results of pMMHl sequencing to develop a technique of food grade vector using the plasmid isolated from Bacillus mesentericus KCTC 0750BP and the other is to search out the portion stable to use as a food grade vector from the constructed vector.
  • To construct vector was carried out as follows:
  • the portions of the pMMHl plasmid were excised to investigate which region is necessary for stable plasmid maintenance with a high copy number.
  • the pMMHl was inserted into the Ncol site within pGEM5zf(+) vector (Promega, U.S.A.) to yield pMMG.
  • Ncol fragments of pMMHl to construct pE ⁇ Ncol fragments of pMMHl to construct pE ⁇ .
  • pE ⁇ was digested with Ndel and then the resulted 4.0kb fragment containing D ⁇ A binding protein coding region, replication origin and secretion protein coding region were ligated into pGEM5zf(+).
  • the D ⁇ A binding protein coding region of pE ⁇ was removed by digestion of pE ⁇ with
  • the pEN without ⁇ -GTP coding sequence showed 98% stability over 100 generations. It suggests that ⁇ -GTP coding sequence is dispensable for plasmid stability.
  • the deletion or disruption of ORF2(pME) or type I signal peptidase in addition(pMN) to ⁇ -GTP coding sequence did not affect plasmid stability significantly.
  • ⁇ -GTP coding sequence, ORE1, ORF2, and type I signal peptidase were deleted but a greater portion of ORFl was deleted in pPS than in pPP. Therefore, the pPS was the smallest vector with the 2.3kb of the pMMHl plasmid.
  • the type I signal peptidase in the pMMHl plasmid is important for stability in Bacillus.
  • Bacillus subtilis has a well-developed extracellular secretion system and type I signal peptidase(type I Spases) plays a main role during secretion and processing of many intracellular molecules through membrane translocase.
  • the type I signal peptidase are located on the chromosomes (SisS, SisT, SisU, SisV and SisW) and plasmid(SisP).
  • SisS and SisP are very important factor for processing and activity of preprotein.
  • SipP is also very important factor because it can functionally replace both damaged SisS and SisT. But, in the example of the prevent invention, the deletion of type I signal peptidase does not affect plasmid stability, suggesting that it is not a necessary factor.
  • the expression vector pJSN was constructed using the smallest and most stable pPS and ⁇ -amylase promoter-signal sequence ( Figure 3).
  • the gene was amplified from pET24-Glul by PCR and cloned at the BamHl and Sail sites of p8Al to construct p8Al- ⁇ 25.
  • the forward primer (5'-GGATCCGAATCTCGCGCTTGAAAGT-3') was designed to contain the BamHl site(underlined) for easy cloning and one nucleotide C(underlined) for the in-frame fusion between the ⁇ -amylase signal sequence and the N-terminus of the ⁇ -glucosidase mature gene.
  • the reverse primer (5'-CTGCAGCTCGA ⁇ TCATCACGCGGT-3') was designed to contain the Xh ⁇ l site(underlined) and random TGA stop codons(underlined).
  • p8Al- ⁇ 25 was digested with EcoRl-HIndl ⁇ l and the 2.5kb-fragment containing ⁇ -amylase promoter-signal sequence- ⁇ - glucosidase were subcloned into pBluescript II KS(+) at the same sites to construct pKS- ⁇ 25. Finally, pKS- ⁇ 25 was digested with Sacll-Apal and then inserted into same sites of the pPS vector to construct the pJSN expression vector. When the oat ⁇ -glucosidase coding gene was inserted into an expression vector, ⁇ -glucosidase was secreted successfully.
  • Fig. 1 is a schematic diagram showing a result that we deleted or disrupted each portion of pMMHl and constructed vector to construct a food grade vector.
  • Fig. 2 is a table listing bacteria and plasmids used in the present invention.
  • Fig. 3 is a schematic diagram showing the pPS vector constructed by comprising only 2.3kb DNA fragment (Pstl-Spe ⁇ ) of pMMHl.
  • Fig. 4 is a schematic diagram of construction of the pJSN vector.
  • Fig. 5 is a schematic diagram of nucleotide and amino acid sequence at fusion site.
  • Fig. 6 is a graph showing growth of cells cultured in a CPGY medium.
  • Fig. 7 is a graph showing ⁇ -glucosidase activity determined by every 3h.
  • Fig. 8 is a photograph showing SDS-PAGE analysis of oat ⁇ - glucosidase activity produced in Bacillus mesentericus DB104 harboring the pJSN vector.
  • EXAMPLE 1 Constructing vector using the pMMHl plasmid derived from Bacillus mesentericus
  • pMMG was based on this, and constructed five vectors (pPS, pPP, pE ⁇ , pM ⁇ and pME) by decreasing the size of the plasmid by deleting or disrupting one by one except replication origin among five regions which pMMHl contained, a replication origin, open reading frame l(ORFl), the ⁇ -GTP, type 1 signal peptidase (sipP) and secretion protein coding region (Fig. 1 and 2).
  • the portions of the pMMHl plasmid were excised to investigate which region is necessary for stable vector maintenance with a high copy number.
  • the pMMHl was inserted into pGEM5zf(+) vector (Promega, U.S.A.) at the Ncol site to yield pMMG.
  • the 1.4kb Sail fragment of the c ⁇ t(chloramphenicol acetyltransferase) gene as a selection marker in Bacillus was cloned into pGEM5zf(+) to construct pGEMC.
  • the EcoRV-Ncol fragment of pGEMC was ligated to the Pvu ⁇ l-Ncol fragment of pMMHl to construct pE ⁇ .
  • pEN was digested with Ndel and the resulted 4.0kb fragment containing DNA binding protein coding region, replication origin and the secretion protein coding region were ligated into pGEM5zf(+).
  • DNA binding protein coding region of pEN was removed by digestion of pEN with EcoRI and then a pM ⁇ was produced by self- ligation.
  • the 2.4kb Pstl fragment and 2.3kb Pstl-Spel fragment containing a replication origin of pMMHl were isolated from pG ⁇ MC and then inserted into the pG ⁇ M5zf(+) to construct pPP and pPS, respectively.
  • 2.3kb DNA fragment (Pstl-Spel) has no problem in stability and includes the smallest portion of pMMHl, we think that it is suited to a food grade vector.
  • the smallest vector is useful as a stable vector because the large size vector is unstable in cells. And when we insert the target gene, the vector becomes so larger unnecessarily that it can't accept the size and lose stability at last.
  • the expression vector pJSN was constructed using the smallest and most stable pPS and ⁇ -amylase promoter-signal sequence (Fig. 4). To place the ⁇ - glucosidase gene under controlling of ⁇ -amylase promoter-signal sequence within vector, the gene was amplified from pET24-Glul by PCR and cloned at the BamHl and Sail sites of p8Al to construct p8Al- ⁇ 25.
  • the forward primer (5'-GGATCCGAATCTCGCGCTTGAAAGT-3') was designed to contain the BamHl site(underlined) for easy cloning and one nucleotide C(underlined) for the in-frame fusion between the ⁇ -amylase sequence and the N-terminus of the ⁇ -glucosidase mature gene.
  • the reverse primer (5 '-CTGCAGCTCGAGTCATCACGCGGT-3 ') was designed to contain the Xhol site(underlined) and random TGA stop codons(underlined).
  • p8Al- ⁇ 25 was digested with EcoRl-HIndl ⁇ l and the
  • pKS- ⁇ 25 2.5kb-fragment containing ⁇ -amylase promoter-signal sequence- ⁇ - glucosidase were subcloned into pBluescript II KS(+) at the same sites to construct pKS- ⁇ 25. Finally, pKS- ⁇ 25 was digested with Sacll-Apal and then inserted into same sites of the pPS vector to construct the pJSN expression vector.
  • a activity of ⁇ -glucosidase was determined by measuring free p- nitrophenol(pNP) from paranitrophenyl- ⁇ -D-glucopyranoside(pNPG).
  • the transformants B. subtilis DB104 harboring the pJSN vector was precultured in 3mL of LB medium containing chloramphenicol(10 ⁇ g/mL) overnight. 3mL of preculture was then transferred to 50mL of
  • CPGY medium in 250 mL-flask and incubated at different growth temperature of 37°C, 30°C, and 25°C with shaking at 200 rpm.
  • One mL of culture broth was centrif ⁇ ged at 15,000 rpm for 20 min.
  • the supernatant of the culture broth(50 ⁇ L) was mixed with 450 ⁇ L of 10 mM pNPG solution resolved in 20 mM potassium phosphate buffer(KH 2 P0 4 , pH5.5). Reaction were carried out at 37°C for 15 min and stopped by addition of a stop solution (2M Na 2 C0 3 solution).
  • the released pNP was determined by reading A 405 .
  • One unit was defined as the amount of enzyme which hydrolyzed lnmol of pNP per min at 37°C.
  • the profile of proteins in supernatant of the culture was analyzed by SDS-PAGE. Proteins in the culture were precipitated adding trichloracetic acid to a final concentration of 10%>(w/v). The precipitates were resuspended in 50 ⁇ L of 0.1 N NaOH. After adding 10 ⁇ L of 5X sample buffer, samples were boiled for 5 min and loaded on 10% SDS- polyacrylamide gel. Because the pPS was stable and the smallest (6.8kb), it was chosen for the construction of a secretion vector. The pPS is the hybrid of the pGEM5zf(+) that is a commercial E.
  • the present invention will be very useful in food and pharmaceutical industries because using food grade vector, pPS which has stability and is easy to produce foreign gene supplies vitamins and proteins etc., things high physiological activity but easy to lack in food.

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Abstract

The present invention relates to food grade vector comprising Pst I-Spe I fragment of pMMH1 plasmid derived from Bacillus mesentericus KCTC 0750BP. The present invention makes it possible to provide vitamin or protein, physiologically active substances needed for food, for introducing the vector to Bacillus living in Korean soy source.

Description

FOOD GRADE VECTOR COMPRISING PST I-SPE I FRAGMENT OF PMMHl PLASMID DERIVED FROM WILD-TYPE BACILLUS
TECHNICAL FIELD
The present invention relates to food grade vector, and more particularly, to food grade vector comprising fragment of pMMHl plasmid derived from wild-type Bacillus mesentericus.
PRIOR ART
Efforts and studies to develop food grade vector have not been going on progress actively, but it has been reported by only Europeans running many diary farms. Because it was not easy to select vectors to do no harm to human bodies and to choose fit selection marker and stable vectors of suitable size, attempts to develop food grade vector have been going on progress passively. These results of previous studies and developments show that it has been difficult to develop food grade vector. Now, food grade vector is developing using plasmid derived from Lactobacillus and Lactococcus lactis, etc. pMMHl is a plasmid isolated from bacteria(5αcz'//«_s' subtilis and its analogues) which has been used for a long time as a major zymogens of the fermentation food made from bean and so whose safety has been recognized internationally and does no harm to human bodies. Based on this advantage, the present invention constructed various vectors using pMMHl to develop a technique of food grade vector, and intended to find the portion comprising the smallest part of pMMHl altogether with having stability among vectors constructed by this method and then here to construct a expression/secretion vector system producing physiologically useful materials.
DISCLOSURE OF THE INVENTION
Therefore, the object of the present invention is to provide edible food grade vector using plasmid derived from wild-type Bacillus which inhabits in kinds of traditional Korean soy source.
The object of the above-mentioned present invention was achieved by isolating the pMMHl plasmid from Bacillus mesentericus KCTC 0750
BP, to confirm that the plasmid was suitable to use as a food grade vector, constructing a vector capable of being used in both Bacillus and E. coli by deleting each portion of pMMHl one by one and ligating E. coli plasmid, pGEM5z(+) to the portion of pMMHl, transforming Bacillus, a host for the constructed vector, culturing it under condition without antibiotics up to 100 generations in successive batch cultures to obtain stability and confirming that 2.3kb fragment^tl-S el) was most suitable to be developed as a food grade vector.
The present invention composes of two steps; the one is to construct vectors by deleting one by one remnant parts except for replication origin among five portions identified by results of pMMHl sequencing to develop a technique of food grade vector using the plasmid isolated from Bacillus mesentericus KCTC 0750BP and the other is to search out the portion stable to use as a food grade vector from the constructed vector. To construct vector was carried out as follows:
The portions of the pMMHl plasmid were excised to investigate which region is necessary for stable plasmid maintenance with a high copy number. For convenient DNA manipulation, the pMMHl was inserted into the Ncol site within pGEM5zf(+) vector (Promega, U.S.A.) to yield pMMG.
Meanwhile, the 1.4kb Sail fragment of the c t(chloramphenicol acetyltransferase) gene as a selection marker in Bacillus was cloned into the Sail site of pGEM5zf(+) to construct pGEMC. The EcoKV-Ncol fragment of pGEMC was ligated to the Pvuϊl-
Ncol fragments of pMMHl to construct pEΝ. To construct pMΝ, pEΝ was digested with Ndel and then the resulted 4.0kb fragment containing DΝA binding protein coding region, replication origin and secretion protein coding region were ligated into pGEM5zf(+). The DΝA binding protein coding region of pEΝ was removed by digestion of pEΝ with
EcoRl and then pME was produced by self-ligation.
Meanwhile, the 2.4kb Pstl-Pstl fragment and 2.3kb Pstl-Spel fragment containing a replication origin of pMMHl were isolated from pGEMC and then inserted into the pGEM5zf(+) to construct pPP and pPS, respectively.
Because rolling-circle replication plasmid, pMMHl separated from Bacillus mesentericus was very stable in a non-selective medium, we tried to develop it into a stable vector in Bacillus. Sequence analysis showed that pMMHl had a replication origin, two open reading frames (ORFs) of the γ-GTP and type 1 signal peptidase(sipP). To characterize the region for plasmid stability, each region was deleted or disrupted in pMMHl. And so five vectors (pPS, pPP, pEN, pMN and pME) were constructed as Bacillus subtilis -E. coli shuttle vectors. The pEN without γ-GTP coding sequence showed 98% stability over 100 generations. It suggests that γ-GTP coding sequence is dispensable for plasmid stability. The deletion or disruption of ORF2(pME) or type I signal peptidase in addition(pMN) to γ-GTP coding sequence did not affect plasmid stability significantly. In both pPP and pPS, γ-GTP coding sequence, ORE1, ORF2, and type I signal peptidase were deleted but a greater portion of ORFl was deleted in pPS than in pPP. Therefore, the pPS was the smallest vector with the 2.3kb of the pMMHl plasmid.
All constructed plasmids were very stable with over 90% stability after about 100 generations. Because γ-GTP coding sequence, ORFl, ORF2 and type I signal peptidase did not aspect plasmid stability significantly, the SSO(single strands origin) between replication origin and ORF2 seems to be most important for plasmid stability.
The type I signal peptidase in the pMMHl plasmid is important for stability in Bacillus. Bacillus subtilis has a well-developed extracellular secretion system and type I signal peptidase(type I Spases) plays a main role during secretion and processing of many intracellular molecules through membrane translocase. The type I signal peptidase are located on the chromosomes (SisS, SisT, SisU, SisV and SisW) and plasmid(SisP). Among these, SisS and SisP are very important factor for processing and activity of preprotein. And, SipP is also very important factor because it can functionally replace both damaged SisS and SisT. But, in the example of the prevent invention, the deletion of type I signal peptidase does not affect plasmid stability, suggesting that it is not a necessary factor.
Therefore, as seeing above, because if the size of vector becomes large, it is unstable in cell, pPS in which unnecessary parts for stable intracellular replication were deleted or disrupted and which included necessary part for stable replication, 2.3 kb DNA fragment (Pstl-Speϊ) of pMMHl is fitter as a food grade vector than any other vectors mentioned.
To conform that a foreign protein was expressed from pPS, the expression vector pJSN was constructed using the smallest and most stable pPS and α-amylase promoter-signal sequence (Figure 3). To place the β- glucosidase gene(1.5kb) under controlling of α-amylase promoter-signal sequence within vector, the gene was amplified from pET24-Glul by PCR and cloned at the BamHl and Sail sites of p8Al to construct p8Al-α25. The forward primer (5'-GGATCCGAATCTCGCGCTTGAAAGT-3') was designed to contain the BamHl site(underlined) for easy cloning and one nucleotide C(underlined) for the in-frame fusion between the α-amylase signal sequence and the N-terminus of the β-glucosidase mature gene. The reverse primer (5'-CTGCAGCTCGAβTCATCACGCGGT-3') was designed to contain the Xhόl site(underlined) and random TGA stop codons(underlined). p8Al-α25 was digested with EcoRl-HIndlϊl and the 2.5kb-fragment containing α-amylase promoter-signal sequence-β- glucosidase were subcloned into pBluescript II KS(+) at the same sites to construct pKS-α25. Finally, pKS-α25 was digested with Sacll-Apal and then inserted into same sites of the pPS vector to construct the pJSN expression vector. When the oat β-glucosidase coding gene was inserted into an expression vector, β-glucosidase was secreted successfully. The activity of β-glucosidase in the supernatant increased steadily after 15h and was the highest at 27h. In SDS-PAGE analysis of proteins in supernatant, the expected β-glucosidase with a size of around 60 kDa was found only in transformants containing an expression/secretion vector, confirming the secretion of β-glucosidase.
The present invention will be explained in more detail with reference to the following examples, but rights of the present invention is not limited to them.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a schematic diagram showing a result that we deleted or disrupted each portion of pMMHl and constructed vector to construct a food grade vector.
Fig. 2 is a table listing bacteria and plasmids used in the present invention.
Fig. 3 is a schematic diagram showing the pPS vector constructed by comprising only 2.3kb DNA fragment (Pstl-Speϊ) of pMMHl.
Fig. 4 is a schematic diagram of construction of the pJSN vector.
Fig. 5 is a schematic diagram of nucleotide and amino acid sequence at fusion site.
Fig. 6 is a graph showing growth of cells cultured in a CPGY medium.
Fig. 7 is a graph showing β-glucosidase activity determined by every 3h. Fig. 8 is a photograph showing SDS-PAGE analysis of oat β- glucosidase activity produced in Bacillus mesentericus DB104 harboring the pJSN vector.
BEST MODES FOR CARRYING OUT THE INVENTION
EXAMPLE 1 : Constructing vector using the pMMHl plasmid derived from Bacillus mesentericus
We cultured Bacillus mesentericus KCTC 0750BP distributed from Korean Research Institute of Bioscience and Biotechnology in LB broth overnight, isolated pMMHl from the cultured solution, digest the plasmid with restriction enzyme, Ncol, digest E.coli plasmid pGEM5zf(+) with same enzyme and then ligated these. We called it pMMG, was based on this, and constructed five vectors (pPS, pPP, pEΝ, pMΝ and pME) by decreasing the size of the plasmid by deleting or disrupting one by one except replication origin among five regions which pMMHl contained, a replication origin, open reading frame l(ORFl), the γ-GTP, type 1 signal peptidase (sipP) and secretion protein coding region (Fig. 1 and 2).
More detailed construction of vector was achieved by the following method:
The portions of the pMMHl plasmid were excised to investigate which region is necessary for stable vector maintenance with a high copy number. For convenient DΝA manipulation, The pMMHl was inserted into pGEM5zf(+) vector (Promega, U.S.A.) at the Ncol site to yield pMMG. The 1.4kb Sail fragment of the cαt(chloramphenicol acetyltransferase) gene as a selection marker in Bacillus was cloned into pGEM5zf(+) to construct pGEMC. The EcoRV-Ncol fragment of pGEMC was ligated to the Pvuϊl-Ncol fragment of pMMHl to construct pEΝ. To construct pMN, pEN was digested with Ndel and the resulted 4.0kb fragment containing DNA binding protein coding region, replication origin and the secretion protein coding region were ligated into pGEM5zf(+). DNA binding protein coding region of pEN was removed by digestion of pEN with EcoRI and then a pMΕ was produced by self- ligation.
Meanwhile, the 2.4kb Pstl fragment and 2.3kb Pstl-Spel fragment containing a replication origin of pMMHl were isolated from pGΕMC and then inserted into the pGΕM5zf(+) to construct pPP and pPS, respectively.
EXAMPLE 2: Searching the most stable portion in constructed vectors
To investigate stability in vectors constructed by above-mentioned example 1 and to search out for a portion we would use as plamid, we transformed Bacillus with the plasmids composed of each portion of pMMHl, inoculated these transformants into 3 mL of LB liquid medium, incubated at 37°C overnight with shaking and then inoculated 2%(1 mL) into 250 mL - triangle-flask including 50 mL of LB liquid broth. And then we transferred them to and cultured in new broth up to 20 generations. And then we transferred them to new pre-warmed broth and then on the same condition cultured in broth without antibiotics up to 100 generations in successive batch cultures. Every 20 generations of growth, the culture was transferred to new broth. After 100 generations, we took about 200 μL among 50 mL of the culture, smeared and cultured overnight. And then about 200 colonies which grew here were transferred to plates containing an antibiotic (chloramphenicol) and were cultured overnight. We confirmed the stability of plasmid through seeing how many bacteria were alive. Constructed vectors had high, above 90% stability(Table 1).
Table 1
The results showing the stability of constructed plasmids based on pMMHl
Figure imgf000011_0001
Because among these, 2.3kb DNA fragment (Pstl-Spel) has no problem in stability and includes the smallest portion of pMMHl, we think that it is suited to a food grade vector. The smallest vector is useful as a stable vector because the large size vector is unstable in cells. And when we insert the target gene, the vector becomes so larger unnecessarily that it can't accept the size and lose stability at last.
Therefore, it need not leaving the unnecessary portions for intracellular stable replication. And after we confirmed stability using the necessary portions for intracellular stable replication, we chosen 2.3 kb Pstl-Spel fragment and inserted it into pGEM5zf(+) to construct pPS the vector(Fig. 3). EXAMPLE 3: Expression of the recombinant genes using constructed vectors
To conform that foreign proteins were expressed through pPS, the expression vector pJSN was constructed using the smallest and most stable pPS and α-amylase promoter-signal sequence (Fig. 4). To place the β- glucosidase gene under controlling of α-amylase promoter-signal sequence within vector, the gene was amplified from pET24-Glul by PCR and cloned at the BamHl and Sail sites of p8Al to construct p8Al-α25. The forward primer (5'-GGATCCGAATCTCGCGCTTGAAAGT-3') was designed to contain the BamHl site(underlined) for easy cloning and one nucleotide C(underlined) for the in-frame fusion between the α-amylase sequence and the N-terminus of the β-glucosidase mature gene. The reverse primer (5 '-CTGCAGCTCGAGTCATCACGCGGT-3 ') was designed to contain the Xhol site(underlined) and random TGA stop codons(underlined). p8Al-α25 was digested with EcoRl-HIndlϊl and the
2.5kb-fragment containing α-amylase promoter-signal sequence-β- glucosidase were subcloned into pBluescript II KS(+) at the same sites to construct pKS-α25. Finally, pKS-α25 was digested with Sacll-Apal and then inserted into same sites of the pPS vector to construct the pJSN expression vector.
A activity of β -glucosidase was determined by measuring free p- nitrophenol(pNP) from paranitrophenyl-β-D-glucopyranoside(pNPG). The transformants B. subtilis DB104 harboring the pJSN vector was precultured in 3mL of LB medium containing chloramphenicol(10 μg/mL) overnight. 3mL of preculture was then transferred to 50mL of
CPGY medium in 250 mL-flask and incubated at different growth temperature of 37°C, 30°C, and 25°C with shaking at 200 rpm. One mL of culture broth was centrifαged at 15,000 rpm for 20 min. The supernatant of the culture broth(50 μL) was mixed with 450 μL of 10 mM pNPG solution resolved in 20 mM potassium phosphate buffer(KH2P04, pH5.5). Reaction were carried out at 37°C for 15 min and stopped by addition of a stop solution (2M Na2C03 solution). The released pNP was determined by reading A405. One unit was defined as the amount of enzyme which hydrolyzed lnmol of pNP per min at 37°C.
The profile of proteins in supernatant of the culture was analyzed by SDS-PAGE. Proteins in the culture were precipitated adding trichloracetic acid to a final concentration of 10%>(w/v). The precipitates were resuspended in 50 μL of 0.1 N NaOH. After adding 10 μL of 5X sample buffer, samples were boiled for 5 min and loaded on 10% SDS- polyacrylamide gel. Because the pPS was stable and the smallest (6.8kb), it was chosen for the construction of a secretion vector. The pPS is the hybrid of the pGEM5zf(+) that is a commercial E. coli cloning vector(Promega, U.S.A.), a portion of pMMHl, and the cαt(chloramphenicol acetyltransferase) gene. Therefore, the pPS functions in both E. coli and B. subtilis. For expression/secretion, an α-amylase promoter-signal sequence system(lkb) of B. subtilis was used. When the oat β-glucosidase gene was inserted, β-glucosidase was secreted successfully. The activity of β- glucosidase in the supernatant increased steadily after 15h and was the highest at 27h(Fig. 6 and 7). In SDS-PAGE analysis of proteins in supernatant, the expected β-glucosidase with a size of around 60 kDa was found only in samples from transformants containing an expression/secretion vector, confirming the secretion of β-glucosidase (Fig. 8).
INDUSTRIAL APPLICABILITY
Hereinbefore, as we described through above-mentioned examples, the present invention will be very useful in food and pharmaceutical industries because using food grade vector, pPS which has stability and is easy to produce foreign gene supplies vitamins and proteins etc., things high physiological activity but easy to lack in food.

Claims

1. A vector comprising 2.3kb fragment obtained by cutting pMMHl derived from Bacillus mesentericus KCTC 0750BP with restriction enzymes, Pstl and Spel.
2. A vector, pPS manufactured by inserting 2.3kb fragment obtained by cutting pMMHl derived from Bacillus mesentericus KCTC 0750BP with restriction ymes, Pstl and Spel into pGEM5zf(+).
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