JP2864285B2 - Lactobacillus-complex shuttle vector for Escherichia coli and host microorganisms transformed therewith - Google Patents

Description

The present invention relates to a novel lactobacillus capable of autonomous growth even in cells heterologous to Escherichia coli lactobacillus and capable of selecting β-galactosidase as the only marker. The present invention relates to a complex shuttle vector for Escherichia coli and a host microorganism transformed thereby.

2. Description of the Related Art Recombinant DNA technology for microorganisms has been developed mainly for Escherichia coli and Bacillus subtilis. In lactic acid bacteria, recombinant DN of lactic acid bacterium
A technology has made considerable progress in the expression of heterologous genes since the establishment of host vector systems.However, it has been difficult to establish a transformation method for lactobacilli, and has a considerable delay compared to other bacterial species. I was

In recent years, however, the successful introduction of the electric pulse method into transformation of lactobacilli, which was almost impossible, has been reported, and several host-hector systems have been established. It became so.

In this lactobacillus host vector system, erythromycin resistance is often used as a selectable marker, and a host vector system in which an erythromycin resistance gene derived from Enterococcus faecalis is integrated on the vector is mainly used. I have. In this system, transformants are effectively selected on media containing the antibiotic erythromycin.

Lactobacillus is a bacterium used for the production of fermented milk, and is a bacterium that has long been confirmed to be safe as a food microorganism. Therefore, this bacterium is improved in breeding by recombinant DNA technology, and by the development of an expression vector,
If useful heterologous genes can be expressed and the production of substances by lactobacilli becomes possible, there is great promise for only those strains that are safe for food use.

However, although the above-mentioned host vector system using erythromycin resistance as a selection marker is an effective system at the laboratory level, it cannot be directly introduced into food production. In this system, since the antibiotic erythromycin is used as a marker for the transformant, if this is used in food production, a large amount of antibiotic is introduced into the food for selection and stable maintenance of the transformant. Because you have to do that.

Therefore, it is necessary to develop a host vector system that does not rely on antibiotic resistance markers and has a selection marker that is safe for use in food production.In Lactobacillus, a host vector system that can be used for food production has been developed. No examples are known.

Problems to be Solved by the Invention The present invention has been made for the purpose of developing a host vector having a selection marker that is safe to use in food production without relying on such an antibiotic resistance marker.

Lactobacillus is mainly used for fermented milk production, in which case,
The medium is milk. Milk contains only lactose as a sugar source. However, since lactobacilli rely on lactose for their energy source,
It cannot grow in milk. Therefore, the lactose-assimilating lactobacillus (β-galactosidase-deficient strain) is used as a host, and a β-galactosidase gene-containing plasmid is used as a vector, whereby β-galactosidase, ie, lactose assimilation, as a selection marker To develop a host vector system that can be used for food production.

Means for Solving the Problems The present inventors have made intensive efforts to develop such a host vector system. As a result, the multiple initiation region (ori) derived from the plasmid pLJ1 of Lactobacillus helveticus, Escherichia coli (Escherichia col.
i) the replication initiation region (ori) derived from plasmid pBR329 and Lactobacillus bulg
aricus) by constructing a complex shuttle vector pBG9 for Lactobacillus-Escherichia coli linked to a β-galactosidase gene derived from a host vector system having a selection marker that is safe for food production without relying on antibiotic resistance markers. Established.

In addition, Enterococcus faecalis (Enterococcus f.) Is located upstream of the β-galactosidase gene of pBG9.
aecalis) by incorporating a region from the promoter of the erythromycin resistance gene derived from the plasmid pAMβ1 to the initiation codon ATG, thereby making this shuttle vector usable as an expression vector and reading through the erythromycin resistance gene from the promoter ( read through), the β-galactosidase gene, which is a selection marker, can be more strongly expressed. In the start codon ATG portion, an appropriate restriction enzyme cleavage site such as a restriction enzyme Sac I cleavage site, a restriction enzyme Aat II cleavage site or a restriction enzyme Sph I cleavage site is provided so that a heterologous gene to be expressed can be inserted. When a heterologous gene is not inserted, the present invention is constructed by constructing a composite shuttle vector by providing a stop codon downstream of the restriction enzyme cleavage site so that unnecessary protein is not produced from the start codon ATG. completed.

That is, the present invention relates to a composite shuttle vector for Lactobacillus-Escherichia coli constructed as described above and a microorganism transformed with this vector.

In the present invention, a composite shuttle vector for Lactobacillus-Escherichia coli which can be used for food production is constructed as follows.

The replication initiation region of Lactobacillus helveticus plasmid pLJ1 is extracted by searching using a restriction enzyme cleavage site present on pLJ1.

The replication initiation region of the plasmid pBR329 of E. coli is pBR329
Utilizing the restriction enzyme cleavage site present above, it is further removed by completely removing the antibiotic resistance gene portion using exonuclease.

For the portion of the erythromycin resistance gene from the promoter to the start codon ATG, insert this gene into the multicloning site of the plasmid vector pUC18 for Escherichia coli, and use this as a template (PCR)
A restriction enzyme cleavage site for insertion of a heterologous gene into the ATG portion, and a stop codon further downstream thereof are synthesized by the oligomer chain reaction method.

In addition, the DN of the chromosome of Lactobacillus bulgaricus
A fragment of the β-galactosidase gene is extracted from A.

The replication initiation region of pLJ1, pBR329 thus obtained and the region from the promoter of the erythromycin resistance gene to the stop codon, as well as the gene fragments of the β-galactosidase gene derived from Lactobacillus bulgaricus, were linked at both ends using a linker. These are ligated by, for example, remodeling to an appropriate restriction enzyme cleavage site to construct a composite shuttle vector.

Lactose assimilating Escherichia coli used as a host in the construction and amplification of this vector includes commercially available Escherichi
a coli JM105 and NM522 are used.

In addition, a lactose non-assimilating host Lactobacillus bacillus is obtained by treating Lactobacillus helveticus without pLJ1 with nitrosoguanidine (NTG) and transforming it with the vector of the present invention.

The microorganism transformed by the composite shuttle vector obtained by the present invention can grow in milk because only the transformant carrying this vector can assimilate lactose, and the vector is dropped off during growth. Bacteria lost due to lactose assimilation are excluded from this system. In this way, it is possible to select a transformant and to stably maintain the transformant without adding any milk, as it is.

In addition, in a certain host microorganism, in order to efficiently express a heterologous gene, the presence of a promoter necessary for the initiation of transcription and the strength of its promoter activity, as well as the nucleotide sequence of the SD sequence upstream of the initiation codon ATG and the initiation codon ATG
Distance to play an important role. In the host lactobacillus Lactobacillus helveticus used in the host vector system of the present invention, it is known that the erythromycin resistance gene derived from the plasmid pAMβ1 of Enterococcus faecalis is efficiently expressed. Therefore, if the portion from the promoter of the erythromycin resistance gene to the initiation codon ATG is used, the heterologous gene can be expressed efficiently in the host lactobacillus. That is, for a heterologous gene having a weak promoter activity, it is directly ligated downstream of the initiation codon ATG. By linking a portion downstream of the start codon ATG of the encoded structural gene to the downstream of the start codon ATG of the erythromycin resistance gene, efficient expression becomes possible. In addition, other cloning sites can be used for a heterologous gene which has no problem in the promoter and the SD sequence and can obtain sufficient expression in the host lactobacillus.

Therefore, the present invention can provide a lactobacillus-Escherichia coli complex shuttle vector that can be used for food production and that enables the expression of a useful heterologous gene in a lactose non-assimilating host lactobacillus.

Next, the present invention will be described with reference to examples (see FIG. 2).

(1) Lactobacillus helveticus plasmid
Acquisition of replication start area (ora) of pLJ1.

There are a number of restriction enzyme cleavage sites on pLJ1. Using this cleavage site, a search was made for the ori portion of pLJ1. As a result, it was found that the ori portion was present on the AvaII-HindIII fragment. This fragment was remodeled into a BamHI-PstI fragment using a linker.

(2) Replication initiation region (or
i) acquisition.

It is known that the ori portion of pBR329 exists between AvaI and PstI. However, the latter part of the antibiotic ampicillin resistance gene exists between Ava I and Pst I. The fact that the antibiotic resistance gene portion is encoded on the vector used for food production is undesirable, so the exonuclease Bal from the Pst I cleavage point
31 was used to completely remove the ampicillin resistance gene. After the removal, the linker was remodeled into a PstI site again using a linker, and the AvaI site was further converted into a salI site so that the ori portion could be removed in the form of a salI-PstI fragment.

(3) Obtaining a portion from the promoter of the erythromycin resistance gene to the start codon ATG.

The erythromycin resistance gene has a Dde size of about 1kb.
It can be excised from pAMβ1 in the form of an I fragment. Both ends of this Dde I fragment were blunt-ended using Klenow enzyme, and inserted into Escherichia coli vector pUC18 which had been similarly blunt-ended after Xba I digestion. Those having the insertion direction as shown in the figure with respect to the multiple cloning site of pUC18 were selected. Synthesis by PCR was performed using Sal I at the multiple cloning site of pUC18.
From the site, the start codon of the erythromycin resistance gene, followed by a new restriction enzyme cleavage site and a stop codon. As a primer for the PCR method, the sequence from the Sal I site was the same as the sequence, and from the start codon, a restriction enzyme Sac I cleavage site was added downstream of the start codon ATG so that it could be used as an expression vector. A stop codon TAA was added downstream of the start codon ATG so as to have the same reading frame as the start codon ATG. That is, G of ATG
And the first G of the Sac I site (GAGCTC) are overlapped with each other, so that this Sac I site is cleaved with the restriction enzyme Sac I and then treated with the Klenow enzyme. It was possible to leave.
As a result, not only a weak promoter activity but also a heterologous gene having a problem with the SD sequence can be expressed by linking a portion of the structural gene downstream of the ATG to the downstream of the ATG. Could be made possible.

Then, a heterologous gene having only a weak promoter activity and having no problem with the SD sequence can be inserted into the Sac I site in the same direction as the promoter of the erythromycin resistance gene as it is.

When a heterologous gene is not inserted at the Sac I site, peptide synthesis from the start codon ATG is stopped by the stop codon TAA provided downstream of the Sac I site, so that the host bacterium has an adverse effect due to unnecessary protein production. I will not receive it.

Using the above two kinds of primers, a DNA containing from the promoter of the erythromycin resistance gene to the start codon ATG, further the Sac I site and the stop codon TAA by the PCR method
Fragments were synthesized.

(4) Acquisition of a β-galactosidase gene derived from Lactobacillus bulgaricus.

The chromosomal DNA of Lactobacillus bulgaricus is cut with the restriction enzyme SalI, and D containing the β-galactosidase gene
Plasmid in which the NA fragment was integrated into the SalI site of pBR329
1 (Japanese Patent Application No. 2-5878).

The plasmid from which the portion (SmaI-SalI portion) downstream of the present gene and which did not encode the present gene was removed from the inserted fragment containing the β-galactosidase gene incorporated into pBG1 was designated as pBG3.

(5) Construction of composite shuttle vector The chloramphenicol resistance gene (Cm ^r ), ampicillin resistance gene (Amp ^r ) of pBG3 obtained in (4) above,
From the BamHI site, including the replication initiation region (ori)
The part up to the I site was replaced with pLJ1 obtained in the previous section (1) and (2).
And the plasmid replaced with the replication initiation region of pBR329
It was set as pBG9.

After pBG9 was digested with SalI and treated with Klenow enzyme, a fragment containing from the erythromycin resistance gene promoter obtained in the above section (3) to the stop codon TAA was inserted into this site. A plasmid in which the direction of the promoter of the erythromycin resistance gene was inserted in the same direction as the direction of the β-galactosidase gene in pBG9 was selected, and this was designated as pBG10. BamH I, Pst I and Sal I sites on pBG10 are available as cloning sites,
The Sac I site can be used as a cloning site for an expression vector.

(6) Lactose assimilating host Lactobacillus helveticus SBT2195 strain was obtained. Lactose assimilating wild-type Lactobacillus helveticus was treated with 300 μg / ml nitrosoguanidine (NTG) in phosphate buffer at 37 ° C. By treating for 30 minutes, a lactose non-assimilating strain was obtained.

The cells after NTG treatment were cultured in an MRS medium (manufactured by DIFCO), and the surviving bacteria were replicated in an MRS medium and a lactose-MRS medium (in which glucose in the MRS medium was replaced with lactose). A strain that grew on the MRS medium but did not grow on the lactose-MRS medium was defined as a lactose non-assimilating strain.

Subculture the lactose non-assimilating strain obtained in this way, select a strain in which the properties of lactose non-assimilating are stable,
This was named Lactobacillus helveticus SBT2195 strain and used as a host lactobacillus.

The SBT2195 strain differs from its parent strain, Lactobacillus, in that it is not lactose-assimilating, but has other mycological properties, such as producing about 90% lactic acid, being gram-soluble, and facultative and aerobic bacilli. Being, catalase (-),
The fact that the lactic acid irradiance was DL type was the same as that of the parent strain Lactobacillus.

The Lactobacillus helveticus SBT2195 strain has been deposited with the Japan Institute of Microorganisms under the deposit number 11538.

Effect of the InventionThe composite shuttle vector for lactobacillus-Escherichia coli of the present invention is capable of autonomous growth even in heterologous cells of Escherichia coli and lactobacillus, and used lactose as a sugar source by combining with a lactose non-assimilating host microorganism This transformant can be selected on a medium.

That is, E. coli E. coli JM105 or E. coli E. coli N
When transformed into M522, the transformant uses lactose as a sugar source
Selection was possible with M9 minimal medium. Further, when the lactose-non-assimilating host Lactobacillus helveticus SBT2195 was transformed, the transformants could be selected on a skim milk medium.

The Lactobacillus helveticus SBT2195 strain transformed with the composite shuttle vector pBG10 of the present invention was named Lactobacillus helveticus SBT2196 strain and deposited with the Microtechnical Laboratory (Deposit No. 11676). SBT2196 strain is pBG10
Holds, it becomes lactose-assimilating like the parent strain.
In addition, other mycological properties, such as about 90% lactic acid
The production, gram-positivity, aeration-anaerobic bacilli, catalase (-), and lactic acid irradiability of DL type were the same as those of the parent strain. When the Lactobacillus helveticus SBT2196 strain is cultured in skim milk, the SBT2195 strain cannot grow at all, whereas the SBT2196 strain can grow and coagulate the skim milk to produce fermented milk. In addition, the SBT2196 strain that has lost the complex shuttle vector due to omission or the like is excluded from this system because it becomes non-assimilate to lactose. That is, the SBT2196 strain can maintain its properties stably only by culturing it in skim milk.

The composite shuttle vector of the present invention is not only useful as a cloning vector, but also has a restriction enzyme cleavage site immediately after the site from the promoter derived from the erythromycin resistance gene to the start codon, so that it is used as an expression vector in lactose bacilli. be able to.

Therefore, the composite shuttle vector of the present invention can be used for food production utilizing these characteristics.

[Brief description of the drawings]

FIG. 1 is a conceptual diagram showing an outline of the composite shuttle vector of the present invention, and FIG. 2 is a conceptual diagram showing an outline of a production method thereof.

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁶ Identification symbol FI C12R 1:19) (C12N 1/21 C12R 1: 225) (56) References Plasmid, Vol. 22 [3] (1989) p. 193-202 Plasmid, Vol. 21 [1] (1989) p. 9-20 (58) Fields surveyed (Int. Cl. ⁶ , DB name) C12N 15/70 C12N 15/74 WPI (DIALOG) BIOSYS (DIALOG) JICST file (JOIS)

Claims (5)

(57) [Claims] 1. The method of claim 1, wherein the Lactobacillus helveticus (Lactob)
replication origin (ori) derived from plasmid pLJ1 of E. acillus helveticus, replication origin (ori) derived from plasmid pBR329 of Escherichia coli, and promoter of erythromycin resistance gene derived from plasmid pAMβ1 of Enterococcus faecalis. Codon ATG, followed by a new restriction enzyme cleavage site, a region containing up to the stop codon, and Lactobacillus bulgaricu
s) The β-galactosidase gene is ligated downstream of a region containing the β-galactosidase gene derived from the erythromycin resistance gene, the initiation codon ATG, the newly established restriction enzyme cleavage site, and the stop codon. A composite shuttle vector for Lactobacillus-Escherichia coli, which is characterized in that: 2. A composite shuttle vector for Lactobacillus-Escherichia coli according to claim 1, which is shown in FIG. 1 and has the following properties:
pBG10. (I) The size is about 6.0 kilobase pairs (kb). (Ii) β-galactosidase can be selected as the only marker. (Iii) (a) A restriction enzyme SacI cleavage site is provided at the start codon ATG of the erythromycin resistance gene. (B) A stop codon is provided downstream of the Sac I site. (C) Expression can be achieved by inserting a heterologous gene into the SacI cleavage site, and when no heterologous gene has been inserted, unnecessary protein production can be stopped by a stop codon. 3. A lactobacillus Lactobacillus helveticus SBT2195 strain, which is a lactose-incapable host lactobacillus of the composite shuttle vector according to (1) or (2). [4] A host microorganism transformed with a complex shuttle vector according to [1] or [2], wherein a lactose non-assimilate lactobacillus is transformed. 5. Lactobacillus helveticus SBT2196
The host microorganism according to claim 4, which is a strain [Deposit No. 11676 of Microtechnical Laboratory].