JPH0527381B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel plasmid containing a DNA fragment containing a gene responsible for the biosynthesis of tryptophan synthase. A DNA fragment containing the gene governing the biosynthesis of tryptophan synthase, such as at least trpA and trpB, and a promoter capable of expressing both of these genes, which is inherited from the parent cell to the daughter cell without being shed during cell proliferation. This invention relates to a plasmid containing a DNA fragment, including a functional DNA fragment.

Various studies have been conducted on the cloning of DNA fragments containing the gene responsible for the biosynthesis of tryptophan synthase.
Journal of General Microbiology, Vol.118,
253 (1980), there are problems with stable retention within the host, and it is believed that there are many difficulties in applying it to the industrial production of tryptophan.

On the other hand, in terms of stable retention of plasmids within a host, the most important problem in practical applications is generally the phenomenon of plasmid shedding during subculturing of the host. Therefore, various attempts have been made to prevent plasmid shedding. For example, a plasmid containing a DNA fragment containing a chromosomal gene that controls the property of being independent of streptomycin in Escherichia species has been used to prevent streptomycin from Escherichia species. A method has been proposed for stabilizing the properties of microorganisms containing plasmids by including them in dependent mutant strains (Japanese Patent Application Laid-open No. 156591/1983).
However, such a method is not only economically problematic, but also requires the incorporation of complex functions into the target plasmid, which makes it difficult to stably distribute the plasmid to daughter cells during host division and multiplication. There are considerable problems in its practical application.

Therefore, the present inventors first aim to stably distribute a DNA fragment containing the tryptophan operon from parent cells to daughter cells over generations, as a DNA fragment containing the gene responsible for the biosynthesis of tryptophan synthase. We conducted research on plasmids that have functions that enable this, including a DNA fragment containing the tryptophan operon, a DNA fragment containing a gene that controls the growth control distribution system of the F factor plasmid, and a DNA fragment that controls the autonomous replication ability of the ColE1-based plasmid. Contains genes
Novel plasmid pMTY-2 containing DNA fragment
(See the specification of Japanese Patent Application No. 59-225906).

Using Escherichia coli carrying this plasmid pMTY-2, it becomes economically possible to produce tryptophan by the so-called fermentation method, but it uses only the tryptophan synthase action derived from the tryptophan operon, and uses indole and L as raw materials. - In the method for producing tryptophan from serine (however, DL-serine may be added to the actual reaction system because only L-serine reacts selectively), tryptophan is In addition to tryptophan synthase, the operon also has the function of coproducing three other enzymes, and if the only purpose is the action of tryptophan synthase, the efficiency will decrease accordingly.

Therefore, in order to improve the efficiency of biosynthesis of tryptophan synthase in the tryptophan operon, the present inventors conducted intensive research to obtain a plasmid suitable for this purpose. As a result, we found that the trpA and trpB fragments, which are the genes responsible for the biosynthesis of tryptophan synthase in the tryptophan operon, have a DNA fragment with a promoter function that can express both genes, and in some cases, a DNA fragment that controls this promoter function. A DNA fragment containing a gene that controls the growth control distribution system of a plasmid derived from the F factor plasmid and a gene that controls the autonomous replication ability of a plasmid derived from a ColE1-based plasmid are combined with a DNA fragment that has a DNA fragment that has an operator function. A plasmid that combines DNA fragments is
The present invention has been completed based on the discovery that the production efficiency of tryptophan synthase can be significantly improved.

Therefore, according to the present invention, at least one of the tryptophan operons derived from Escherichia coli
A DNA fragment containing the trpA and trpB genes, a DNA fragment with a promoter function that can express both genes, a DNA fragment with an operator function that can control the promoter function, and proliferation derived from the F factor plasmid. A mini-F fragment containing the gene governing the regulatory distribution system,
A recombinant plasmid characterized by containing a DNA fragment containing a gene that controls the ability to autonomously reproduce derived from a ColE1-based plasmid, and an Escherichia coli K-12 microorganism transformed with the recombinant plasmid. provided.

DNA containing the gene responsible for the biosynthesis of tryptophan synthase that constitutes the plasmid of the present invention
Fragment” (hereinafter sometimes abbreviated as “T fragment”)
is indole and L- or DL-serine to L
-Contains genes that control tryptophan biosynthesis
The T fragment used in the present invention, which refers to a DNA fragment, includes:
In particular, trpA, a gene that controls the biosynthesis of tryptophan synthase in the tryptophan operon.
and trpB, a DNA fragment with a promoter function that can express both genes (hereinafter sometimes referred to as "tryptophan promoter") and a DNA fragment with an operator function that can restrict the promoter function as necessary ( This includes DNA fragments bound with a tryptophan operator (hereinafter sometimes referred to as a "tryptophan operator"). As such a T fragment, one derived from Escherichia coli is preferably used practically. There are no particular restrictions on the source of this T fragment, but Escherichia coli
ATCC23282, Escherichia coli ATCC23437,
Escherichia coli ATCC 23461 and the like are advantageously used.

From these source microorganisms
trpA and trpB fragments combined with tryptophan promoter and tryptophan operator
The detailed method for preparing the DNA fragment is shown in (B) of Example 1 below, but the basic procedure is to infect E. coli, which has a tryptophan operon in its chromosomal gene, with phage φ80, and then induce it. fuage
A large amount of phage incorporating the tryptophan operon into DNA is prepared. Next, Fage
Extract the DNA, cut out the tryptophan operon DNA fragment using restriction enzymes BamH, EcoR, etc., and further cut out the tryptophan operon DNA fragment using restriction enzymes Hinc
Partial cleavage was performed to contain the trpA and trpB genes.
DNA fragments are obtained. In addition, when the tryptophan operon DNA fragment is treated with the restriction enzyme Pru,
A DNA fragment containing the promoter and operator is obtained. A DNA fragment in which the trpA and trpB fragments are bound to a tryptophan promoter and a tryptophan operator can be prepared by joining both of the obtained DNA fragments with a DNA ligation enzyme.

As described above, the present invention allows the above T fragment to be converted to F
One feature is that it is combined with a DNA fragment containing a gene that controls the growth control distribution system of a plasmid derived from a factor plasmid. For example, the F factor plasmid is described in "Protein Nucleic Acid Enzyme" Vol. 27 No. 1 (1982)
It has a structure as shown in the genetic map in Figure 1 on page 98 and the physical map by Eco R, and the molecular weight is
It is a known plasmid of 94.5 kb (62 × 10 ⁶ Daltons), which is normally present in enterobacteria such as Escherichia coli at a copy number of 1 to 2 per cell chromosome, and this plasmid is transferred to each daughter cell after cell division. (In this way,
A mechanism for keeping the copy number at a low level and increasing it precisely at the same pace as the host's proliferation.
This is called strict growth control). Such a stringent growth control function in the F factor plasmid is caused by a 9.1 kb molecular weight protein called mini-F.
It has already been determined that mini-F is carried by a fragment that can autonomously reproduce, and it is also known that this mini-F can be excised from the F factor plasmid by the regulatory enzyme EcoR.

The present invention utilizes the growth control distribution system carried by this mini-F, and therefore uses the "DNA fragment containing the gene governing the growth control distribution system derived from the F factor plasmid" (hereinafter referred to as "F fragment"). (sometimes abbreviated) means a gene fraction equipped with a mechanism for accurately transmitting the F factor plasmid to daughter cells as described above, and a typical example of such an F fragment has a molecular weight of approximately 9.1 kb. An example is the mini-F fragment having the following.

Furthermore, in the present invention, the "DNA fragment containing a gene that controls autonomous replication derived from ColE1 plasmid" (hereinafter sometimes abbreviated as "S fragment") used in combination with the above T fragment and F fragment is a It refers to a DNA fragment containing a gene that controls the autonomous replication of ColE1-based plasmids, which number 20 to 30 per cell chromosome, and a typical example of such an S fragment is a plasmid derived from pBR322, which has a length of approximately 4.3 kb. In addition, the S fragment of plasmid
There is an S fragment derived from pBR325 etc.

The plasmid provided by the present invention has three essential components: T fragment, F fragment, and S fragment described above.
As long as it has DNA fragments, it carries other genetic information
It may further include a DNA fragment, such as a DNA fragment containing an ampicillin resistance gene, which is an antibiotic resistance marker, a DNA fragment containing a kanamycin resistance gene, etc., and one typical example is a T fragment, a F
It consists essentially of three DNA fragments: a fragment and an S fragment, and has a molecular weight of approximately 9.0 megadaltons (approximately 13.6 kb).
The present inventors have developed a plasmid called “Plasmid”.
pMTY-3". In addition, in this specification, the molecular weight of a plasmid is a value measured by agarose gel electrophoresis.

This plasmid pMTY-3 will be explained in more detail below.

The sensitivity (number of recognition sites) of the following restriction enzymes of plasmid pMTY-3 and the length (kb) of fragments degraded by the restriction enzymes are shown in the table below.

Number of fragments digested at recognition site restriction enzyme position (kb) EcoR 2 10.7, 2.9 BamH 2 6.9, 6.7 Sal 3 10.6, 2.9, 0.1 Pst 5 5.9, 4.0, 1.8, 1.5, 0.4 Ava 1 13.6 As mentioned above The plasmid pMTY-3 of the present invention having such characteristics can be produced, for example, as follows.

First, a DNA fragment (T fragment) in which a tryptophan promoter and a tryptophan operator are linked to trpA and trpB fragments is prepared by, for example,
Escherichia coli that has a tryptophan operon in its chromosomal gene, such as Escherichia coli K-12
(IFO3301, ATCC10798, ATCCe23562), etc., for example, Phage φ80
(ATCC11456a-B1) etc., and by utilizing lysogenization and induction phenomena, we prepared a large amount of phages incorporating the tryptophan operon into the phage DNA [RMDenney, C. Yanofsky; J.
Bacteriol., 118 , 505 (1974)], then the conventional method (EFFritsch, Sambrook,
“Molecular cloning” (1982) p.164-165, Cold
Extract the phage DNA using restriction enzymes such as BamH (see Spring Harbor Laboratory).
, tryptophan operon using EcoR etc.
Cut out a DNA fragment, further partially cleave this DNA fragment with the regulatory enzyme Hinc, and generate trpA and trpB.
A DNA fragment containing the gene is obtained, and when the tryptophan operon DNA fragment is treated with the restriction enzyme Pvu, it contains the promoter and operator.
DNA fragments are obtained. Next, both of the obtained DNA fragments were joined together using T4 DNA ligase derived from T4 phage.
Furthermore, by mixing an EcoR linker and ligating with T4 DNA ligase, the DNA fragment containing the promoter and operator is bound to the trpA and trpB fragments, yielding a T fragment having EcoR sites at both ends.

On the other hand, the mini-F fragment can be prepared using microorganisms carrying the F factor plasmid, such as E. coli K-12 strain (ATCC15153,
ATCCe 23589, ATCCe 23590) etc. in a manner known per se, for example from P. Guerry, DL. Le Blanc
The F factor plasmid was extracted by the method described in the literature such as S. Falkow; J. Bact., 116 , 1064 (1973),
The restriction enzyme EcoR is then used to reduce the molecular weight to approx.
It can be prepared by cutting out a 9.1 kb mini-F fragment.

On the other hand, ColE1 is the source of the DNA fragment containing the gene that controls autonomous replication of the ColE1 plasmid.
Representative plasmid pBR322 as a plasmid
It is convenient to use.

The mini-F fragment prepared as above was treated with restriction enzymes EcoR and BamH, and combined with plasmid pBR322 treated with the same restriction enzymes.
Plasmids are made using T4 DNA ligase.
A plasmid in which the above mini-F fragment is integrated into pBR322 is created. This plasmid is then
The desired plasmid pMTY-3 can be obtained by cleaving it with EcoR, combining it with the T fragment prepared as described above, and ligating it with T4 DNA ligase.

A specific method for preparing plasmid pMTY-3 will be described in more detail in Example 1 below.

The plasmid of the present invention prepared in this way has a large number of copies and has the excellent properties of being stable and less likely to be shed when passed on to daughter cells during cell division of the host.

Therefore, it is highly expected that the plasmid of the present invention will be industrially applied in the production of tryptophan. For the production of tryptophan, a host is transformed with the plasmid of the present invention. As a host bacterium that can be used for this transformation, E. coli is preferable, and a tryptophanase-deficient mutant strain of the host bacterium is particularly preferable.

In addition, the plasmid of the present invention can be introduced into these host bacteria using methods known per se, such as M.
Mandel, A.Higa; J.Mol.Biol. 53 , 159 (1970)
This can be carried out by the method described in the literature such as .

The thus-transformed host bacteria is cultured by a method known per se to sufficiently produce and accumulate tryptophan synthase within the cells, and then L- or DL-serine is converted into L- or DL-serine.
- Can be used in enzymatic reactions when producing tryptophan.

When using cultured bacterial cells for the enzyme reaction,
The bacterial cells can be used as they are, but they can also be used as a crushed product obtained by crushing the bacterial cells by ultrasonication, an extract obtained by further extracting the crushed material with water, or the extract further extracted with ammonium sulfate, etc. It can also be used in the form of a crudely purified product obtained by treatment to precipitate the enzyme component, and furthermore, the cells or the treated product can also be used after being immobilized, if necessary.

The reaction between indole and L- or DL-serine in the presence of the bacterial cells or their processed material can be carried out using, for example, a 0.1M phosphate buffer (PH7.0~
9.0) or in a solvent such as water (PH7.0-9.0), approx.
The reaction is usually carried out at a temperature of 20 to about 50°C, preferably about 30 to about 40°C, for about 10 to about 72 hours.

Although there is no particular restriction on the amount of indole and L- or DL-serine used in the reaction, it is generally appropriate to use each in a concentration range of 0.1 to 20% (wt/vol). Further, there is no particular restriction on the amount of the bacterial cells or the processed product thereof, but it can generally be used at a concentration of 1 to 10% (wt/vol).

The culture of the above-mentioned transformed bacterium varies depending on the type of host bacterium, but in general, it can be carried out using commonly used synthetic or natural media. As a carbon source, various carbohydrates such as glucose, glycerol, fructose, sucrose, and molasses can be used. Further, as the nitrogen source, natural organic nitrogen sources such as tryptone, yeast extract, corn steep liquor, and casein hydrolyzate can be used. Many natural organic nitrogen sources can serve as carbon sources as well as nitrogen sources.

Cultivation can be carried out under aerobic conditions such as shaking culture or submerged culture with aeration. The culture temperature is generally 20 to 50°C, and the pH of the culture medium is preferably maintained near neutral or slightly alkaline. The culture period is usually 1 to 5 days.

Indole and L-, or
The separation and purification of L-tryptophan produced in the reaction solution obtained by reacting DL-serine is as follows:
This can be carried out by known methods such as adsorption and desorption treatment using ion exchange resins, activated carbon, etc.

Furthermore, host bacteria transformed with the plasmid of the present invention can also be used to produce L-tryptophan by fermentation. That is, if a host transformed with the plasmid of the present invention is cultured in a medium containing indole, L-tryptophan will be produced and accumulated in the medium, and L-tryptophan can be produced by collecting this.

Example 1 Construction of plasmid pMTY-3 (A) Preparation of phage φ80pt Escherichia coli (E. coli) K-12 strain (IFO3301) was
ml of L medium (Bacto tryptone 10g, yeast extract 5g, NaCl 5g, glycose 1g, water 1
; PH7.2) and shaken at 37℃ for about 4 hours, and phage φ80
(ATCC11456a-B1) aqueous solution (10 ⁵ /ml)
0.1ml and L medium soft agar (L medium + agar drop)
After mixing, layer it on an L medium agar plate. When the plate is incubated at 37°C for about 5 hours, plaques (lytic plaques) are formed, and 2 to 3
When the culture is continued at 37°C for several days, growing colonies of Phage φ80 lysogens are generated in the plaques.
After culturing the lysogenic bacteria in L medium at 37°C for 4 hours, they were spread on the same L medium agar plate as above, and then irradiated with ultraviolet light (400 to 800 ergs/mm ² , 10 to 20
Phage φ80pt (phage DNA containing the tryptophan operon) is prepared by induction of lysogenic phages by (Sec).

(B) Preparation of T fragment in which tryptophan promoter and operator DNA fragments are linked to trpA and trpB fragments E. coli K-12 strain (IFO3301)
of L medium (crude is the same as above), and approx.
Culture with shaking at 37℃ for about 3 hours, and 25% in logarithmic growth phase.
Add 10 ml of (w/v) glucose solution and the Phage φ80pt solution prepared above at a concentration of 10 ^{to 11} cells/ml (moi 20), continue shaking for 5 hours, and then add chloroform in the usual manner to prepare a large amount of Phage φ80pt. Prepared [T. Maniatis, EFFritsch,
Sambrook; “Molecular cloning” (1982)
See p.76-80 Cold Spring Harbor Laboratory].

Next, the obtained Phage φ80pt solution was dialyzed against Tris buffer (PH7.8), and then DNA extraction was performed using the phenol method [see "Molecular Cloning" above].
The phage DNA was extracted and purified using [see p.
Incubate for tryptophan operon DNA
Got a piece.

Next, 10 μg of this tryptophan operon DNA fragment was partially treated with the restriction enzyme Hinc at 37°C for 5 minutes to obtain DNA containing the trpA and trpB genes.
2 μg of fragment was prepared. Further, 20 μg of the tryptophan operon DNA fragment was treated with restriction enzyme Pvu at 37° C. for 1 hour to prepare 0.4 μg of a DNA fragment containing the promoter and operator gene regions. Mix both obtained DNA fragments
They were bound by reacting at 12°C for 24 hours using T4 DNA ligase, further mixed with 0.5 μg of EcoR linker, and recombined by reacting at 12°C for 24 hours using T4 DNA ligase. Furthermore, this recombinant was treated with the restriction enzyme EcoR at 37°C for 1 hour.
The promoter and operator DNA fragments are bound to the trpA and trpB fragments, and both sticky ends are
A T fragment with an EcoR site was prepared.

(C) Construction of plasmid pMTY-3 In order to construct plasmid pMTY-3 according to the present invention, first, as shown in the attached FIG.
Construct pBR322-miniF plasmid.

The mini-F fragment was prepared using a conventional method [Mukai, T.
Matsubara, K., Takagi.Y: Proc. Natl.
Acad.Sci.USA, 70 2884 (1973)], and 1 μg of this mini-F fragment is treated with the restriction enzyme BamH at 37° C. for 1 hour. On the other hand,
Separately prepared Amp ^r (ampicillin resistance) and
Tc ^r (tetracycline resistance) plasmid
Restriction enzyme EcoR and
Let BamH act for 1 hour at 37°C. After that, both treated products were mixed and T4 DNA ligase was added at 12℃.
was allowed to act for 24 hours to recombine. after recombination
The DNA was transformed into Escherichia coli C600 strain (ATCCe23738), and a strain having both Amp ^r and Tc ^s (tetracycline sensitivity) was selected. Plasmid DNA was isolated and purified from these strains, and its molecular weight was measured by agarose gel electrophoresis.
A 10.7 kb pBR322-miniF plasmid was collected.

Next, as shown in Figure 2, pBR322−miniF
pMTY-3 is constructed by ligating the plasmid with the T fragment prepared in (B) above.

i.e. pBR322−miniF plasmid
After treating 2 μg of DNA with restriction enzyme EcoR at 37°C for 1 hour, trpA and
Promoter and operator in trpB fragment
Mix 1μg of DNA fragments bound together,
Religion was performed using T4 DNA ligase at 12°C for 24 hours. Using the recombined DNA, Escherichia coli K-12 strain (tryptophan auxotrophic mutant, ATCC23718) was transformed according to a conventional method, and the transformed strain [loss of Trp auxotrophy; that is, trpA and trpB genes on the plasmid] was transformed using the recombined DNA. Tryptophan biosynthesis is possible with minimal medium (K ₂ HPO ₄ 7g, KH ₂ PO ₄ 2g, MgSO ₄ .
7H ₂ O 0.1g, (NH ₄ ) ₂ SO ₄ 1g, glycose 2
g. Bacterial strains that can grow on pure water 1)]
I got it. This strain was cultured in liquid according to a conventional method, and plasmid pMTY-3 having the restriction enzyme map shown in FIG. 2 was isolated and purified from the culture solution.

The transformed strain carrying this plasmid pMTY-3 was stored as Escherichia coli K12 YK2009 at the Institute of Microbial Technology, Agency of Industrial Science and Technology, 1-1-3 Higashi, Yatabe-cho, Tsukuba-gun, Ibaraki Prefecture, in 1982.
The microorganism has been deposited under the FERM Article No. 7957 (FERM P-7957) on November 27, 1991. Submission No. 3244 (FERM
It has been transferred to the International Deposit as BP-3244].

Example 2 Stability of transformed strain 100 ml of the above minimal medium was dispensed into a 500 ml Erlenmeyer flask, sterilized at 120°C for 15 minutes, and the transformed strain obtained in Example 1 was inoculated into the flask. After 24 hours of shaking culture at ℃, L
Dispense 100ml of culture medium into a 500ml Erlenmeyer flask and heat to 120°C.
The cells were sterilized for 15 minutes and subcultured at a rate of 50 cells per ml, and cultured with shaking at 37°C for 24 hours. Next, collect bacteria using centrifugation,
After washing the bacterial cells, a certain amount of the cells is plated on a plate medium prepared as an L medium supplemented with ampicillin at a rate of 50 μg/ml or an L medium without the addition of ampicillin, and grown colonies are counted after culturing at 37° C. for 1 day.

As a result, it was confirmed that the number of colonies grown in ampicillin-added and non-ampicillin-added media was the same, and that L
It was confirmed that all medium-grown colonies grew on a minimal medium containing no tryptophan, that is, the high stability of the plasmid was confirmed.

Example 3 Measurement of tryptophan synthase activity 100 ml of minimal medium was dispensed into a 500 ml Erlenmeyer flask, sterilized at 120°C for 15 minutes, and the transformed strain obtained in Example 1 was inoculated, and the flask was incubated at 37°C. After doubling with shaking for one day, add 20 ml to L medium containing similarly prepared indole acrylic acid at a concentration of 100 μg/ml.
The cells were inoculated and cultured with shaking at 37°C for 6 hours. Collecting bacterial cells by centrifuging the culture solution,
Wash with 50ml of 100mM Tris buffer (PH7.8),
After centrifugation was performed again to collect the bacteria, 200 mg of wet bacterial cells were collected, suspended in 1 ml of 100 mM Tris buffer (PH7.8), and subjected to ultrasonication. After treatment, the crushed bacterial cells were appropriately diluted with 100mM Tris buffer and subjected to the conventional method [O.
H. Smith and C. Yanofsky: “Methods in
Enzymology”, Academic, NawYork (1962),
vol 5, p 794-806].
As a result, when E. coli (FERM BP-3244) carrying the plasmid pMTY-3 of the present invention was used, approximately 250 units (1 unit = 0.1 μmoleTrp/mg
The activity value of protein/20min) is shown. Furthermore, when the activity was examined by the above method using plasmid pMTY-2 as a control, it showed an activity value of about 100 units.

[Brief explanation of the drawing]

Figure 1 shows the construction process of plasmid pBR322-miniF, and Figure 2 shows plasmid pMTY.
It is a figure showing the creation process of -3.

Claims (1)

[Scope of Claims] 1. A DNA fragment containing at least the trpA and trpB genes in the tryptophan operon derived from Escherichia coli, a DNA fragment having a promoter function capable of expressing both of these genes, and a DNA fragment capable of controlling this promoter function. A DNA fragment to which a DNA fragment with an operator function has been combined, a mini-F fragment containing a gene governing the growth control distribution system derived from the F factor plasmid, and a DNA fragment containing a gene governing autonomous proliferation ability derived from the ColE1-based plasmid. A recombinant plasmid characterized by comprising: 2. A DNA fragment containing at least the trpA and trpB genes in the tryptophan operon derived from Escherichia coli, a DNA fragment having a promoter function capable of expressing both of these genes, and a DNA fragment having an operator function capable of controlling this promoter function. This is a recombinant plasmid containing a DNA fragment in which F factor plasmid is derived, a mini-F fragment containing a gene governing the growth control distribution system derived from the F factor plasmid, and a DNA fragment containing a gene governing autonomous replication ability derived from the ColE1 plasmid. A transformed Escherichia coli K-12 microorganism.