JPH03285677A - Culture of transformed microorganism - Google Patents

Abstract

PURPOSE:To enable efficient manifestation of the objective structural gene by culturing a transformed microorganism containing a specific DNA fragment in a tryptophan-containing medium while adding a specific amount of glucose to the medium. CONSTITUTION:A microorganism such as Escherichia coli YK3007 (FERM BP-2803) is transformed with a recombinant plasmid pMTP4, etc., at least containing (A) a DNA fragment containing a promoter in tryptophanase operon (tna) and a regulator gene tnaC positioned at the downstream of the promoter and (B) a DNA fragment containing the objective structure gene mainifestable by the promoter. The microorganism is cultured under aerobic condition in a medium containing L- or DL-tryptophan while continuously or intermittently adding glucose as a carbon source at a rate to keep the glucose concentration to 0.01-0.3%(wt/vol). The objective structure gene can be manifested by this process to enable the production of useful substances in high yield.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention involves culturing a microorganism transformed with a recombinant plasmid containing a desired structural gene derived from an animal, plant, or microorganism, which can be expressed by a promoter in the tryptophanase operon. The present invention relates to a method for efficiently producing a large amount of the expression product of a target structural gene, a novel recombinant plasmid that can be used in the method, and a microorganism transformed with the plasmid.

In recent years, new genetic recombination techniques have been developed that use a host microorganism that carries a complex plasmid containing a gene fraction that carries the genetic information for useful substances derived from animals, plants, or microorganisms, and allow the microorganisms to produce large quantities of the useful substances mentioned above. Remarkably, production of insulin, interferon, etc. is already underway using Escherichia coli as a host microorganism.

However, although genetic recombination technology has advanced as described above, a method for mass-producing the desired product using genetically modified bacteria that possesses the desired structural gene has not yet been established, and the efficient use of genetically modified bacteria There is an urgent need to develop a culture method.

On the other hand, the gene expression mechanism of the tryptophanase operon is controlled by an expression regulation mechanism called "catabolite repression" (BoLsford J,
L, and R, D, DeMoss; J.
Bac.

It is known that the expression of this gene is extremely strongly suppressed in a medium containing glucose as the main carbon source.

Therefore, although the promoter in the tryptophanase operon has the ability to express the target structural gene, it is necessary to cultivate a microorganism carrying a plasmid containing the target structural gene expressed by the promoter and the promoter. When attempting to cause the microorganism to express the target structural gene at a high level, it is not possible to use a medium containing glucose as the main carbon source, and it is necessary to use a medium containing succinic acid or other expensive carbon sources. I was forced to do it.

In this way, in order to express useful structural genes using the strong gene expression ability of the promoter in the tryptophanase operon, it is possible to use a cheap and industrial method as the main carbon source when culturing microorganisms carrying such genes. The inability to use glucose, which is also widely used, is a major obstacle to the industrial use of this promoter.

The present inventors discovered that the tryptophanase operon (tna)
Focusing on the powerful gene expression mechanism of the promoter, we conducted intensive research on its effective use. As a result, we found that the promoter in the tryptophanase operon (tna) and the regulatory gene t located downstream of the promoter
When the DNA fragment (b) containing the target structural gene is linked to the downstream side of the DNA fragment (a) containing naC, not only the tryptophanase structural gene but also the tryptophanase structural gene is generated by the promoter.
A foreign structural gene of interest was also found to be strongly expressed.

Moreover, a microorganism transformed with a recombinant plasmid containing at least the above DNA fragments (a) and (b),
As mentioned above, despite being subjected to catabolite repression (suppression of gene expression by glucose), the concentration of glucose in the culture solution containing L- or DL-tryptophan is limited to a certain low level. When cultured with continuous or intermittent addition to maintain within this range, surprisingly, catabolite repression does not occur, the target structural gene is highly expressed, and the expression product of the gene is It has been found that it is possible to obtain high yields.

Note that Japanese Patent Publication No. 61-42555 describes a method of culturing a microbial medium by continuously or intermittently adding glucose at a low concentration to the medium, in which the glucose concentration in the medium is kept at 1% or less. A method for culturing the tryptophan synthase-producing strain E 5 Cherichia coli has already been disclosed, which comprises adding glucose continuously or intermittently so as to increase the concentration of the tryptophan synthase.

However, the expression of the tryptophan synthase structural gene is controlled by the bromotar in the l-lyptophan operon, and this gene expression mechanism is catabolic).
It is known to receive no represions.

In fact, Agr. Biol. Chem., 38 (
7), 1335-1342. In 1974, the tryptophan synthase-producing bacterium Escherichia coli (Escherichia coli)
The tryptophan synthase activity of cells cultured at 0.5% and 1.8% glucose concentrations in the culture medium was 2.4% and 1.8%, respectively.
and 7.3 U/m+2, and the enzyme activity increases with increasing glucose concentration, and JP-A-612681
Publication No. 75 discloses that tryptophan synthase activity is highest when the tryptophan synthase-producing bacterium Escherichia coli is cultured while maintaining the glucose concentration in the medium at 0.5 to 5%. .

In the above-mentioned Japanese Patent Publication No. 61-42555, in the examples, the glucose concentration in the medium was set to 1% and 0, respectively.
．． When cultured at a glucose concentration of 0.05%, the cell yield was 2.69 times higher and the enzyme activity was 1.80 times higher at a glucose concentration of 1% than when cultured in a medium containing 5% glucose at the start of culture. Double increase and glucose concentration 0.05%
-c 3.1 times the bacterial cell yield and 2.15 times the enzyme activity
It has been disclosed that the amount will increase twice as much. From this result, it can be seen that there is a tendency for bacterial cell yield and enzyme activity to increase at lower concentrations of gluco-7, but the differences are small. Furthermore, there is no report that the tryptophanase operon used in this culture method is a recombinant DNA strain capable of expressing the tryp I fan synthase structural gene.

Furthermore, when gene expression is suppressed by catabolite repression, only gene expression is suppressed, regardless of bacterial cell proliferation, but as mentioned above, Escherichia coli used in Japanese Patent Publication No. 61-42555 In this case, both bacterial cell yield and enzyme activity are suppressed by glucose.

For these reasons, the phenomenon disclosed in Japanese Patent Publication No. 61-42555 has a completely different mechanism from catabolite repression, and only Escherichia coli, the L-lyptophan synthase producing bacterium used in the method, This is considered to be a very specific phenomenon.

As described above, methods for culturing the tryptophan synthase-producing bacterium Escherichia coli have been disclosed, in which glucose in the medium is maintained at various concentrations.

However, as far as the present inventors are aware, there has been no report regarding an industrial culture method for a transformed Escherichia coli strain using the tryptophanase operontor gene expression mechanism of the present invention. Thus, according to the present invention, a DNA fragment (a) comprising a promoter in tryptophanase oberon (tna) and a regulatory gene tnaC located downstream of the promoter, and a DNA fragment (a) comprising a target structural gene expressible by the promoter.
A microorganism transformed with a recombinant plasmid containing at least fragment (b) is transformed into L- or DL-IJ.
In a medium containing butophane, glucose is used as a carbon source at a concentration of 0.01 to 0.3% (wt/vo).
The transformed microorganism is cultured while being continuously or intermittently added to the medium so as to be maintained within the range of Q), thereby causing the microorganism to express the target structural gene. A method of culturing microorganisms is provided.

Hereinafter, the culture method according to the present invention will be explained in more detail.

(2) Transformed microorganism The microorganism cultured according to the method of the present invention contains (a) a promoter in the tryptophanase operon (Lna) and a regulatory gene located downstream of the promoter. na
(b) a DNA fragment containing a target structural gene that can be expressed by the promoter (hereinafter referred to as a "T fragment"); A microorganism transformed with a recombinant plasmid containing at least the following: This transformed microorganism will be described below.

[-1. Recombinant plasmid" 2. The recombinant plasmid introduced into the host microorganism when preparing a transformed microorganism contains the above DNA fragment (a), that is, the P fragment, and the DNA fragment (b), that is, the T fragment. There are no particular restrictions on the DNA fragments, as long as they contain a limited amount of DNA, are stably retained and replicated in the host microorganism, and may contain any DNA fragment of an appropriate capacity.

The above-mentioned P fragment has the function of expressing the target structural gene that is desired to be expressed by incorporating it into the plasmid, and the chromosomal DNA of Escherichia coli is practically suitable as its source. . Specifically, for example, Escherichia coli ATCC
27325 Esierhia coli ATCC 2328
2. Chromosomal DNA of strains such as Escherichia coli ATCC 23437 and Escherichia coli ATCC 23461 can be advantageously used.

Specific methods for preparing P fragments from these source microorganisms are described in Example 1, (1) and Example 2, (2) below, but the basic operating method will be briefly described. Chromosomal DNA containing trypto-7 anase oberon was obtained from a culture of the source microorganism Escherichia coli using a conventional method [5aito H, and Miura, K, B
iochemica B 1ophysica Act
a 72 619-629 (1963)], the chromosomal DNA is cut with an appropriate restriction enzyme, and a DNA fragment containing the promoter and regulatory gene tnaC in trypto7 anoberon (tna) is cloned. Thus, for example, the restriction enzymes BamHI and Hin
If excised using dlII, a tryptophanase operon (tna) DNA fragment with a length of about 3.2 kb can be obtained, which is further digested with restriction enzymes AluI and Rsa.
If excised using I, a DNA fragment containing the tna promoter and the regulatory gene tnaC with a length of about 630 bp can be prepared, and this DNA fragment can be used as the P fragment.

In addition, if the target structural gene in the T fragment is the tryptophanase structural gene (tnaA), the above-mentioned tryptophanase (tna) DNA fragment with a length of approximately 3.2 kb or the BamH1 site of this DNA fragment Approximately 900b
The length of p deleted with oxonuclease is approximately 2.3k.
A DNA in which the DNA fragment b is linked with a P fragment and a T fragment
Can be used as fragments.

On the other hand, the "target structural gene" contained in the T fragment refers to a structural gene derived from microorganisms, animals, or plants that carries genetic information corresponding to proteins, peptides, etc. that can be expressed by the P fragment.

Typical examples of useful substances carried by such target structural genes as genetic information are as follows.

I. liptophanase (E, C, 4, 1, 99, 1),
Tryptophan synthase (E, C, 4, 2, I, 2
0), chloramphenicol acedertransferase (EXC, 2, 3, 1, 28), aspartase (E, C14, 3, 111), threonine synthase (E, C, 4, 2, 99, 2) ), homoserine kinase (E, C, 2, 7, 1, 39), various growth hormones, insulin, somatosfutin, 1 2 interferon, interleukin, etc.

DNA fragments (T fragments) containing the target structural genes that carry the genetic information for these useful substances are obtained from cells of animals, plants, or microorganisms capable of producing the useful substances using commonly used methods known per se. can be obtained. In the present invention, those derived from Escherichia coli are particularly preferably used.

Preferred specific examples of the T fragment include a DNA fragment containing the tryptophanase structural gene (tnaA) and the tryptophan synthase structural gene (trpA 1trpB).
Examples include DNA fragments containing Q. In addition, these DNA
Specifically, the microorganism that serves as a source of fragments is E.
5 Cherichia coli K-12 strains, such as Escherichia coli ATCC27325, Escherichia coli ATCC23282, Escherichia coli
ATCC23437, Escherichia coli ATCC2
3461 etc. can be used.

The DNA fragment containing the tryptophanase structural gene (tnaA) is a tryptophanase operon (tna) DNA with a length of approximately 3.2 kb, which is excised from the Escherichia coli chromosomal DNA using restriction enzymes BamHI and HindIff. Fragment and its DNA
An example is a DNA fragment with a length of about 2.3 kb obtained by deleting about 900 bp from the BamH1 site of the fragment. As described above, these are DNA fragments in which the tryptophanase structural gene (tnaA) is linked downstream of the P fragment, and can be used as a DNA fragment containing both the P fragment and the T fragment.

In addition, the tryptophan synthase structural gene (trp
A, trpB) can be obtained by, for example, infecting the chromosomal DNA of E. coli with phage 〆80 and then inducing it, preparing a large amount of phage that incorporates the tryptophan operon into the phage DNA, Next, the 7age DNA was extracted and the tryptophan operon DNA with a length of about 20 kb was extracted using the restriction enzyme BamHI.
Cut out the A fragment and further digest this DNA fragment with restriction enzyme H.
1 with a length of about 2.6 kb obtained by performing a partial needle 3 4 solution with 4ncI[
- Ributophane synthase structural gene ([rpA,
trpB)-containing DNA fragments.

Furthermore, other DNA fragments that may be included in the recombinant plasmid according to the present invention include a DNA fragment containing a gene that controls the ability of C01E1-based plasmids to autonomously reproduce, a DNA fragment that contains a gene that controls the growth control distribution system derived from the F factor plasmid, and a DNA fragment that contains a gene that controls the proliferation control distribution system derived from the F factor plasmid. Examples include DNA fragments containing genes responsible for resistance to substances (eg, ampicillin, kanamycin, talolamphenicol, etc.).

Therefore, one of the preferred recombinant plasmids in the present invention is
Specific examples include the following four DNA fragments constructed by the present inventors: (a) a DNA fragment containing the promoter in the t-lyptophanase operon (tna) and the regulatory gene tnaC located downstream of the promoter; (P fragment); (b) A DNA fragment (T fragment) containing the target structural gene that can be expressed by the above promoter: (c) Col
D containing the gene that governs the autonomous replication ability of El-based plasmids
NA fragment: (d) A plasmid carrying a DNA fragment containing a gene controlling the growth control distribution system derived from the F factor plasmid can be mentioned.

When constructing this plasmid, the above DNA fragment (c) used in combination with the P fragment and the T fragment, that is, the "DNA fragment containing the gene responsible for autonomous replication derived from the Co1El system plasmid" (hereinafter referred to as "S (sometimes abbreviated as "fragments") have a copy number of 20 copies per chromosome per cell.
It includes DNA fragments containing ~30 genes that govern autonomous replication of Co1El-based plasmids. Such S
Typical examples of fragments include, for example, the S fragment derived from plasmid pBR322 having a length of approximately 4.3 kb or the S fragment derived from plasmid pKK2328 having a length of approximately 5.1 kb; The derived S fragment can be exemplified.

The 5 6 P fragment, T fragment and S fragment are further combined with a DNA fragment (d) containing a gene governing the growth control distribution system of the plasmid derived from the F factor plasmid. For example, the F factor plasmid is based on the genetic map shown in Figure 1 on page 98 of "Proteins, Nucleic Acids, Enzymes", Vol. 27, No. 1 (1982) and EcoR.
It is a known plasmid with a molecular weight of 94°5 kb (f32XlO' Daltons), with a structure as shown in the physical map by I. present, and this plasmid has a mechanism that allows it to be transmitted precisely into each daughter cell after cell division (thus keeping the copy number at a low level and precisely paced with host growth). The mechanism that increases the number of cells is called stringenL growth control). Such st in the F factor plasmid
It has already been determined that the controlling function of ringent proliferation is carried out by a fragment called m1ni-F, which has a length of 9.2 kt and can reproduce autonomously.
r & General genetics” I 9
6.185-193 (1984)], this m1n
It is also known that i-F can be excised from the F factor plasmid using the restriction enzyme EcoRI.

The plasmid according to the present invention utilizes the growth control distribution system carried by m1ni-F, and is therefore a "DNA fragment containing a gene controlling the growth control distribution system derived from the F factor plasmid" (hereinafter referred to as "F (sometimes abbreviated as "fragment") refers to the gene fraction responsible for the mechanism for accurately transmitting the F factor plasmid to daughter cells as described above. Representative examples of such F fragments include E. coli. K
] The m1ni-F fragment with a length of about 9.2 kb is excised with the restriction enzyme EcoR,I from the F factor plasmid that can be obtained from 25train.

The plasmids that can be used in the present invention include the above-mentioned P
Four essential DNA fragments: T fragment, S fragment and F fragment
As long as it has a DNA fragment that carries other genetic information, such as a DNA fragment that contains the ampicillin resistance gene, which is an antibiotic resistance marker, or a DNA fragment that contains the kanamycin resistance gene.
A fragment etc. may be further contained.

The construction of recombinant plasmids from such DNA fragments can be carried out in methods known per se, such as those described in standard texts on genetic engineering techniques [eg T, Maniatis E, F.

Frisch and J, Sambrook
“Mo1eculear Cl.

ning” Co1d Spring Harbor
(1982) et al.].

Typical examples of recombinant plasmids that can be used in the present invention include those consisting essentially of four DNA fragments: P fragment, T fragment, S fragment, and F fragment, in which (1) the T fragment is Tryptophanase structural gene (Lna
A) A plasmid with a molecular weight of approximately 10.2 megadaltons (approximately 15.5 kb), which is a DNA fragment containing A), which the present inventors named "plasmid pMTP4J" (see Example 4 below) (2) T fragment is the tryptophanase structural gene ([na
Plasmids pMTP1 and pMTPIR with a molecular weight of approximately 10.7 megadalin (approximately 16.4 kb), which are DNA fragments containing A) (these plasmids and their preparation method are disclosed in JP-A-63-28393). Because there are
(3) The T fragment is the tryptophan synthase structural gene (
trpB, trpA) with a molecular weight of approximately 9.9 megadaltons (approximately 16°6 kb).
(see Example 2 below), among which plasmid pMTP4 and plasmid pMTY20 are preferred. Furthermore, the above-mentioned plasmid pMTP4 and plasmid pMTY20 are novel plasmids that have not been described in conventional literature, and constitute a part of the present invention.

Plasmid pMTP4 has the number of recognition sites for restriction enzymes and the length of fragments degraded by the restriction enzymes (as described in Cloth 1 below).
kb).

0 Table 1 Plasmid pMTP4 Recognition site Length of degradation fragment Number of restriction enzymes (k b) EcoRI 2 9.2, 6.3B
amHT 3 2.410.
7.2.4 HindlI[214,7,0,8 Plasmid pMTP4 with the characteristics described above is
For example, it can be manufactured as follows.

First, the preparation of a DNA fragment ((P t T) fragment) in which the tryptophanase promoter and the regulatory gene tnaC/ribtophanase structural gene 11aA located downstream of this promoter is combined is carried out, for example, when tryptophanase is present in the chromosomal gene. Escherichia coli with an operon, such as Escherichia coli strain 112 (ATCC232
82, ATCC23437, ATCC27325, AT
CC23461), etc., and then extracted using a conventional method [E, F, Fr1Lsch, Sambr
ook, ”Molecular cloning
(] 982) p, 164-165, Cold
A pring Harbor Laborato
Restriction enzymes BamHI and Hind
When the tryptophanase operon DNA fragment is excised using I[[, a DNA fragment of approximately 3.2 kb is obtained.

Next, 900 bp was deleted from the BamH1 site of the fragment to create an approximately 2.3 kb fragment containing the trizo]fanase promoter, the regulatory gene tnaC located downstream of this promoter, and the tryptophanase structural gene tnaA.
PtT) fragment is obtained.

On the other hand, the regulation of the m1ni-F fragment can be carried out using microorganisms carrying the F factor plasmid, such as E. coli K-12.
Strains (ATCC15153, ATCC23282, AT
CCe23590), etc., in a manner known per se, for example P, Guerry D.

L, La Blanc, S, Falkow; J
, Bact, 1-C'-11064 (197
It can be prepared by extracting the F factor plasmid by the method described in the literature such as 3) and cutting out the m1niF fragment with a molecular weight of about 9.2 kb using the restriction enzyme EcoRff.

On the other hand, it is convenient to use plasmid pBR322, which is a typical Co1El plasmid, as a source of a DNA fragment containing a gene that controls autonomous replication of the Co1El plasmid.

The (PtT) fragment prepared as described in Section 1 above was combined with plasmid pBR322 treated with restriction enzymes BamHI and HindII, and treated with T4 DNA ligase to add the above (PtT) to plasmid pBR322.
A plasmid into which the fragment has been integrated is created. This plasmid is then cleaved with the restriction enzyme EcoRI, combined with the m1niF fragment prepared as above, and T4
By applying DNA ligase, the desired plasmid pMTP4 can be obtained.

A specific method for preparing plasmid pMTP4 will be described in more detail in Example 1 below.

On the other hand, plasmid pMTY20 shows the number of restriction enzyme recognition sites and the length (kb) of fragments degraded by the restriction enzyme as shown in Table 2 below.

Table 2 Plasmid pMTY2Q EcoRI 2 9.2.7.
4HindlI[39,3,5,0,2,3SalI
4 12.8.2.4.0.8.0
．． 6BamHI 5 6.8.4
．． 0.2.7.2.4.0.7 Plasmid p M TM0 with the characteristics described above
1 can be manufactured, for example, as follows.

First, DNA containing the tryptophanase promoter and the regulatory gene tnaC located downstream of the plasmid
The fragment (P fragment) can be prepared by, for example, Escherichia coli having a tryptophanase operon in its chromosomal gene, such as Escherichia coli strain ATCC 23282, ATCC 23282, AT
CC23437, ATCC27325, ATCC234
Chromosomal DNA was extracted from 61, etc., and then extracted using the standard method [E
,F.

Fritsch, Sambrook, “M.
o1ular cloning (1982) p. 1
64-165, Cold Sprin3 4g Harbor Laboratory], cut out the tryptophanaseoberon DNA fragment using the restriction enzymes BamHI and HindI, and then cut out the tryptophanase promoter of about 630 bp and the tryptophanase promoter using the restriction enzymes Rsa I and AluI. A DNA fragment containing the regulatory gene LnaC located downstream of this promoter is obtained. Furthermore, a P fragment can be obtained by adding a BamHI linker.

In addition, DN containing the tryptophan synthase structural gene
The A fragment (T fragment) can be prepared by injecting Escherichia coli, such as Escherichia coli strain 112 (ATCC 27325, ATC
C23282, ATCC23437, ATCC2346
1), etc., 7age, such as Phage〆80 (AT
CC11455a-Bl) was infected, and using lysogenization and induction phenomena, Trizol-7 was added to the phage DNA.
A large amount of 7age incorporating the amp operon was prepared [R
, M., Denny, C., Yanofsky; J.
, Bacteriol,, l ] 8.505
(1974) Reference 1, then the conventional method [E, F,
Fritsch, Sambrook.

”Mo1ecular cloning (]
982) p, I 64-165, Cold
Spring Harbor I-aborator
Phage D is extracted according to [see y], a tryptophan operon DNA fragment is excised using restriction enzymes such as BamHI, 5EcoRI, etc., and this DNA fragment is further partially digested with restriction enzyme HincII to obtain a DNA fragment containing the trpA and trpB genes. can get.

Next, the obtained DNA fragment and 5af2 I linker are mixed and ligated using T4 DNA ligase to obtain a T fragment having 5a12 I sites at both ends of the trpA and LrpB fragments.

On the other hand, the m1ni-F fragment (F fragment) can be prepared by using a microorganism carrying the F factor plasmid, such as Escherichia coli -12 strain (ATCCl 5153,
ATCCe23589, ATCCc23590) etc. in a manner known per se, for example P.

Guerry, D. L., LeB 1anc, S.
, Falkow; J.

The F factor plasmid was extracted by the method described in the literature 5 6 such as Back.
It can be prepared by cutting out the 1ni-F fragment.

On the other hand, as a source of the DNA fragment (S fragment) containing the gene that controls the autonomous replication of the Co1El plasmid, Co1E
It is convenient to use the representative plasmid PBF322 as the I plasmid.

After inserting the P fragment and T fragment prepared as above into the BamHI site and the 5alI site of plasmid pUcI]9 (manufactured by Takara Shuzo), Ec again.

The tryptophanase promoter and regulatory gene tnaC and the tributophantase structural gene (trpA,
A fraction containing trpB) is obtained. Obtained D
The NA fragment was converted into Ec of plasmid pBR322 (S fragment).
inserted into the oRI and Hind111 sites, and further inserted into the EcoR
By inserting the F section into the I site, the desired plasmid pMTY20 can be obtained.

The specific method for preparing plasmid I) MTY20 will be described in more detail in Example 2 below.

1-2. Host microorganism The host microorganism that can be transformed with the recombinant plasmid described in the previous section I-1 is EScler.
Microorganisms belonging to ichia coli are preferred, but
Depending on the type of the plasmid, for example, P seudomonas puLida
), Brevibacterium flavum (Brevib
Microorganisms such as Bacterium f 1aVum and Bacillus subtilis may also potentially be used as hosts.

Examples of microorganisms belonging to Escherichia coli include:
Escherichia coli strain 112 (ATCC27325
, ATCC23282, ATCC23437, ATCC
23461 etc.) can be used.

I-3. Transformation The recombinant plasmid described in the previous section i-1 and the hos described in the previous section 78 1-2 can be introduced into microorganisms by methods known per se, for example, M., Mandel, A.
.

Higa; J, Mo1. Biol...
53.159 (1970) and the like.

Specific examples of microorganisms transformed in this manner include the following.

(1) Escherichia coli YK3007 (FERM BP-2803) carrying the plasmid pMTP4
) (2) Escherichia coli harboring the plasmid pMTP112YK3002 (FERM BP-
1733), and Escherichia coli YK3003 (FERMBP-1734) carrying the plasmid pMTPIR (3) Escherichia coli YK2017 (FERMBP-2804) carrying the plasmid pMTY20 The traits described in (1) to (3) above The transformed Escherichia coli strain has been deposited at the Ministry of International Trade and Industry, Microbial Technology Research Institute, 1-1-3, Tsukuba South East, Ibaraki, under the accession number in parentheses above.

(2) Culture The transformed microorganism according to the present invention may be cultured by L- or DL-
In a medium containing tryptophan, glucose is used as a carbon source at a concentration of 0. O1~0.3% (wt/
The medium is continuously or intermittently added to the medium so as to be maintained within the range of vol. As a result, the transformed microorganism does not undergo catabolite repression,
The gene expression mechanism of the promoter in the tridi-1-fanase operene (tna) works strongly, and the target structural gene is expressed to produce useful substances at high yields.

The glucose concentration in the medium was 0.01 to 0.0 during the culture period.
3% (wt/vol), preferably 0.02-0.2
5% (wL/vol), more preferably 0.03~
0.2% (wt/vol), thereby making it possible to omit the use of other industrially expensive carbon sources. However, in some cases other carbon sources may be used, such as glycerol, succinic acid,
Acetic acid, fumaric acid, malic acid, etc. may also be used.

In addition, the culture medium used in the present invention includes L- or D L
- Tryptophan is added.

This allows the transformed microorganism to strongly activate the gene expression mechanism of the promoter in the tryptophanase operon (tna).

The concentration of Shi- or DL-1-lyptophan in the medium is not strictly limited, but is preferably 0.1-2.
% (wt/vol) More preferably 0°05 to 0.5
% (wL/vol). L or DL-tryptophan may be added at the early or middle stage of the culture, but is preferably added before the late stage of logarithmic growth.

Furthermore, nutrients such as nitrogen sources, inorganic salts, growth promoting substances, etc. are appropriately added to the culture medium used in the present invention, depending on the type of microorganism to be cultured. Examples of nitrogen sources that can be included in the medium include ammonium salts such as ammonium sulfate, ammonium chloride, ammonium nitrate, and ammonium phosphate; nitrates such as potassium nitrate, sodium nitrate, and ammonium nitrate; organic nitrogen such as glutamic acid, glutamine, aspartic acid, and asparagine; Examples include ammonia, and these can be used alone or in combination. Examples of inorganic salts include potassium hydrogen phosphate,
Potassium dihydrogen phosphate, magnesium sulfate, iron sulfate, manganese sulfate, and the like can be used alone or in combination. Further, growth promoting substances include, but are not particularly limited to, vitamins such as thiamine and biotin; amino acids such as methionine and cysteine; or yeast extracts containing all or part of these;
Examples include polypeptone, meat extract, cornstarch liquor, and casamino acids.

The content of nutrients that can be included in these media is not particularly limited and can be the content used for normal culture, but nutrients derived from natural products are particularly important for economic efficiency and bacterial cell growth. Due to the influence of the above, it is appropriate that the content is usually in the range of 1 to 0.1%, preferably 0.5 to 0°I 2 2%.

Cultivation of microorganisms in a medium having the composition as described above can be carried out under aerobic conditions using the same culture capacity as normally used. In particular, by maintaining the dissolved oxygen concentration in the medium generally in the range 1 to 8 ppm, preferably 2 to '7 ppm, by aerating and/or agitating the medium with air or other enzyme-containing gas, Cultivation can be performed more efficiently.

Further, glucose may be added to the culture solution continuously, or may be added at regular intervals or intermittently while monitoring the glucose concentration.

Here, the "dissolved enzyme concentration" during culture can be measured using, for example, a dissolved enzyme electrode, and the "glucose concentration" can be measured using, for example, Glucose C Test Fuco (manufactured by Wako Pure Chemical Industries, Ltd.) or Glucose Analyzer GLU-1 (Toa This can be done using a radio wave product.

Cultivation conditions can be selected depending on the type of transformed microorganism used, etc., but, from a practical perspective, the culture temperature is about 10 to about 450 C1, preferably about 2
The temperature can be in the range of 5 to about 40°C, and the pH of the medium can be in the range of about 3 to about 10°, preferably about 5 to about 9. If the pH of the medium changes during culture, it is desirable to adjust the pH by appropriately adding alkalis such as ammonia, caustic soda, caustic potash, sodium carbonate, and sodium bicarbonate, or acids such as sulfuric acid and hydrochloric acid. The culture time can usually be about 5 to about 48 hours.

By culturing as described above, a significant amount of the useful substance carried by the target structural gene can be accumulated within the microorganism or in the culture solution.

The useful substance can be recovered from the microbial cells or the culture solution by a method known per se, for example, centrifugation, ultrafiltration, or the like.

Next, the present invention will be explained in more detail with reference to Examples. However, the following examples are given only to help gain a concrete understanding of the present invention, and the scope of the present invention is not limited in any way by these examples.

In addition, in the Examples, (1) the number of recognition sites by restriction enzymes and the size of cleavage fragments, (2) tryptophanase activity, (3) l-lyptophan synthase activity, (4) taloramphenicol acetyltransferase ( C.A.T.
) Activity, (5) amount of protein and glucose in the test enzyme solution, (6) amount of recovered bacterial cells, etc. were measured by the following methods.

(1) Number of recognition sites by restriction enzymes and size of cleavage fragments The "number of recognition sites" by restriction enzymes means that a DNA fragment or plasmid is completely degraded in the presence of an excess of restriction enzymes, and these degraded products are themselves It can be determined from the number of separable fragments subjected to 1% agarose gel electrophoresis and polyacrylamide gel electrophoresis according to known methods.

In addition, when using agarose gel electrophoresis, the "cut fragment size" and the plasmid size are determined by the DNA of Escherichia coli lambda phage (λphage).
Based on the standard line drawn by the migration distance on the same agarose gel of a DNA fragment of known molecular weight obtained by cutting with the restriction enzyme Hind1m, and when using polyacrylamide gel electrophoresis, the Escherichia coli file・X 174 Phage (I x 1.74 ph
The size of each DNA fragment of the cut DNA fragment or plasmid is determined based on the standard line drawn by the migration distance of the DNA fragment of known molecular weight obtained by cutting the DNA of the plasmid (Age) with the restriction enzyme Haell on the same polyacrylamide gel. Calculate. The size of the plasmid is determined by adding the sizes of each cut fragment. In addition, in determining the size of each DNA fragment, regarding the size of fragments larger than lkb,
Adopting the results obtained by 1% agarose gel electrophoresis, approximately 0. For the size of fragments smaller than 1kb], the results obtained by 4% polyacrylamide gel electrophoresis were used.

(2) Tryptophanase activity Centrifugation (4000 RPM) from 20 ml of culture solution.

The cells were suspended in 100 mmol phosphoric acid buffer (pH 8,0) 20+nQ and then centrifuged again (4000 RPM, 15 minutes, 4°C) to collect the cells. After suspending the bacterial mass in the above buffer solution 2mα, it was subjected to ultrasonic disruption treatment (Sonifier 200 manufactured by Branson), and the treated material was centrifuged (12000 RPM for 140 minutes,
4°C) The soybean supernatant liquid was used as the test enzyme solution.

For the reaction, the above enzyme solution was mixed with 100 mmol phosphate buffer (
After diluting an appropriate amount at pH 8,0), 0.1ml of the diluted solution
and the reaction solution [10.9 ma of 100 mmol phosphate buffer (pH 8°0) containing 10 mmol of L-tryptophan and 0.04 mmol of pyridoxal phosphate at 37°C. The shaking reaction is carried out in a constant temperature water bath for 20 minutes. The reaction was carried out with 1 m of 10 wt% trichloroacetic acid.
(After adding 2 and stopping, centrifugation (4000PPM, 1
The amount of indole produced in the supernatant obtained at 40C for 5 minutes was quantified by gas chromatography (GC-3BF, manufactured by Shimadzu).

The activity of tryptophanase is 1 per unit protein amount.
It was calculated as the amount of indole produced per hour, and expressed as a relative value with the activity of the comparative example as 1.

(3) Tryptophan synthase Centrifugation (4000 RPM) from 20 ml of culture solution.

Suspend the bacterial cells in 20ml of lOOmM l.lis hydrochloric acid buffer (pH 7, 8) (20ml) and centrifuge again (4000 RPM, 15 minutes, 4°C) to collect the bacterial cells. After suspending the collected bacteria in the above buffer solution 2m+2, ultrasonic disruption treatment (Branson Sonifier 200) was performed, and the supernatant liquid obtained by centrifuging the treated material (1200 ORPM, 40 minutes, 4°O) This was used as a test enzyme solution.

To measure the enzyme activity, use the Yanofs enzyme solution as usual.
[Method in Ezymol
ogy, vol. 5.794 (1962) reference 1.

Tryptophan synthase activity was calculated by calculating the amount of indole consumed per hour per unit amount of protein, and expressed as a relative value with the activity of the comparative example set as 1.

(4) Collect the bacterial cells by centrifuging the chloramphenicol acetyltransferase (CAT) activity culture solution, and
After washing with 50 mQ of M Tris buffer (pH 78) and centrifuging again to collect bacteria, collect 200 mg of wet bacterial cells.
+Q 10mM) was suspended in Liss buffer (pH 7, 8) and subjected to ultrasonication. To measure the enzyme activity, the treated bacterial cell fragments were appropriately diluted with 10 ml of Lys buffer.
W, V as usual.

The method of Shaw et al. [Method in Enzym
ology, Academic Press, Ne
w York (1975), vol 43, p 73
7]. The chloramphenicol acetyltransferase activity was calculated as the amount of chloramphenicol acetylated per minute per unit protein amount, and expressed as a relative value with the activity of the comparative example as 1.

(5) The amount of protein and glucose in the test enzyme solution was measured by the method of Lawry et al. (Journal of
B i.

Logical Chemistry, vol.
93, p. 265.1951) and the measurement of glucose concentration in the medium was performed using the Glucose C Test Wako [
manufactured by Wako Pure Chemical Industries, Ltd.].

(6) Amount of recovered bacterial cells Culture solution 50m (2 to centrifugation (8000 rpm, 2
Measured as the amount of wet bacterial cells collected after 0 minutes at 4°C.
I.

(1) In the trizo] fanase operon (tna),
Preparation of a DNA fragment containing the promoter, the regulatory gene tnaC located downstream of the promoter, and the tryptophanase structural gene (tnaA) Medium (10 g of tryptone, 5 g of yeast extract, 1 g of glucose, NaC
Dispense 15 g of distilled water lQ: pH 7, 2) 100 mff into a 500 m4 Erlenmeyer flask, and heat at 120°C for 15 minutes.
Sterilize for minutes.

Escherichia coli (E 5c11eric) was added to this medium.
hiacoli) K-12ATCC27325 and cultured at 37°C for 15 hours, 290 ml of this culture was taken and inoculated into 100 mQ of the above medium, and cultured again at 37°C for 4 hours. I did this.

After culturing, the entire volume of the culture solution was centrifuged (8000 x 9
．． Collect bacteria for 15 minutes at 4°C, and reduce bacterial cells to 50 mM
hLis buffer (pH 8,0)-10mMEDTA・2
Suspended in 50ml of Na solution (2).Next, lysotium was added to a final concentration of 2ml/m4, and after standing for 5 minutes, 6m4 of 10% sodium dodecyl sulfate solution was added, and the suspension was heated at 65°C for 30 minutes. This lysate was kept warm.5M
Add 15 mQ of NaCl solution, cool at 0°C for 1 hour, and centrifuge the entire volume (12000Xz, 60 minutes, 4
°O) L, separate the upper groove fraction, add twice the volume of ethanol, mix, and centrifuge (5000Xy, 10 minutes, 4
°C). LomrvI the obtained precipitate! JS buffer (pH 7,5) ImM EDTA ・2Na
DN is dissolved in a solution, treated with phenol (removal of protein) and treated with RNA degrading enzyme, and finally DN
1.5 mg of A was obtained.

25 μg of the chromosomal DNA prepared as above was treated with restriction enzymes HindI[l and BamHI (5 units each).
The chromosomal DNA was cut by reaction at 30°C for 11 hours using
A solution of H1ndll and BamHI decomposition product was prepared. Plasmid pBR3221μ9 was added to this digest solution with restriction enzymes Hindl11 and BamHI (each) uni
ts) at 30°O for 1 hour.
), l0mM dithiothreitol, lmM ATP, l
Add each component of OmM MgCI 2 and 1 unit of T4 ligase (concentration of each component is final limit), 16
React for 15 hours at °C to allow binding.

Using this solution, a conventional method [M, Mandel, A.

Higa; J, Mo1. Biol,, 5
3.159 (1970)].
Coli (E 5cherichia coli) K 1
Two strains (tryptophanase-deficient mutant and tryptophan auxotrophic mutant) were transformed, and selective medium (K2HP
0.7g, KH2PO42g, (NHI)zsOalg
, Mg5O, 7H200, 1g, casamino acid 5g, Indoheru 10mg, adenine 50mg, Koba 1Ie29
, 20 g of agar, and pure water (IQ). This medium' second growth strain was placed on L medium, and ansibilin was added at a final concentration of 50! I
After inoculating into a medium containing g/ma and culturing at 37°C for 7 hours, the bacterial cells were centrifuged (8000X9.10 minutes at 4°C).
) collected by. Tryptophanase activity was examined using this bacterial cell.

Tryptophanase M phosphate buffer pH 8.0, lOml VI tryptophan, O.M. 1% Triton X-100) Jiff with 5 wet bacterial cells
0 mg was added and reacted at 37°C for 30 minutes.

When the produced indole was colored using p-dimethylbenzaldehyde, a red colored strain was obtained. From this strain, the alkali=SDS method [T. Mania
tis. E., F., Fricsch and
J.

Sambrook; ”Molecular clone
ning (1982) p90-91]
The plasmid was extracted with BamHI and HindI, and the molecular weight was examined using agarose gel.
Approximately 3.2 kb of DNA was inserted into the I[ and BamHT sites (hereinafter, this plasmid will be referred to as pB R 3 2
2 tna).

The transformed strain obtained above was cultured in liquid according to a conventional method, and the plasmid pB R 3 2 2 tnan was extracted from the culture solution.
Mira refined. The plasmid DNA was treated with the restriction enzyme BamH.
After complete decomposition by reacting with I and HindIII at 37°C for 1 hour, the decomposed product was separated by 0.8% agarose gel electrophoresis, and contained a tryptophanase structural gene of approximately 3.2 kb. A DNA fragment fraction was cut out from the gel, and the DNA was extracted and purified to obtain a DNA fragment. Next, this DNA fragment was introduced into plasmid pUC119, and p
UC119tna was produced as follows.

Plasmid pUCll9 is a plasmid of approximately 3.1k that is replicable in Escherichia coli and expresses ancipillin resistance.
It is a plasmid of b and can be purchased from Takara Shuzo as a commercial product.

0.5μ9 of the above plasmid pUc119 was reacted with restriction enzymes BamHI and HindI[l at 37°C for 1 hour to completely degrade it. BamHI, H containing the decomposition product and the above 3 4 tryptophanase structural genes.
After mixing the indlTI DNA fragment fractions and heating at 65°C for 10 minutes to inactivate the restriction enzyme, the components in the inactivation solution were added to Liss buffer pH 7.6 (final concentration of 50 mM each). , 10mM MgCI2, IOmM dithiothreitol, ImMATP and T4 ligase lun
Each component was added so as to give the desired temperature, and the mixture was kept at 16°C for 15 hours. Escherichia coli JM1 using this solution
09 competent cells (manufactured by Takara Shuzo) were transformed.

The transformed strain was treated with 50 μg/mQ (final concentration) of Ansibilin 100 pg/m (2 (final concentration) IPTG (isopropyl-β-D-thiogalactopyranoside), l 0 0
pq/mf2 (final concentration) containing X-gal (5-bromo-4-chloro-3indolyl-β-D galactopyranoside) (tryptone log, 5 g of yeast extract)
NaCl 51? , pure water Iff.

A viable strain was obtained by culturing at 37° C. for 24 hours (pH 7.2). Among these growing strains, those that grew as white colonies were selected, and each plasmid was added to alkaline-S
DS method [T, ManiatlSsE.

F., Fritsch, J., Samb.
rook, ”Molecular cloning
(1982) p90-91] (hereinafter this plasmid is referred to as pUclI9tna).

Obtained plasmid pUC l l 9tna DNA
using the restriction enzymes BamHI and Bbel at 37°
c. Completely decomposed after reacting for 1 hour. The unnecessary part of about 900 bp from the BamHI site of this cleavage fragment was deleted using exo or flase ■ (manufactured by Takara Shuzo), and Bbe
The portion (2) was treated with 51 screase (manufactured by Takara Shuzo) to make it a blunt end. Next, BamHI linker (manufactured by Takara Shuzo) was mixed (50mM)! JS buffer (pH 7, 6), 10
mMMgC1. and each component of T4 DNA ligase lunit (the concentration of each component is the final concentration), 16
Plasmids were reconstituted by ligation by reaction at °C for 15 hours.

Using this plasmid solution, a conventional method [M. Mandel
,A. Higa; J, Mo1. Biol. ,
Escherichia coli 56 (E 5chcrichia cadi) K-12 strain (tryptophanase-deficient mutant and tryptophan auxotrophic mutant) was transformed according to [5 3, 159 (] 970)], and the selective medium (K2HPO4
7g, KH2PO(2g, (NHh)2S
It was smeared on O4i g, Mg5ot.7H200, Ig, casamino acid 5g, indole 10mg, adenine 50mg, glucos 2g, agar 20g, pure water 112).

The strains grown on this medium were cultured in liquid according to a conventional method, plasmid DNA was extracted from the culture solution, and restriction enzyme BamH
I and Hindm, reacted at 37 °C for 1 hour,
After complete decomposition, they were separated by 08% agarose gel electrophoresis, and approximately 2.9 kb (pUC119 portion) and approximately 2.3 kb (pUC119 portion) were separated.
The one showing kb (tna part) was selected. Furthermore, this 2.3 kb DNA fragment was excised from the gel, the DNA was extracted and purified, and the tryptophanase promoter, regulatory gene LnaC, and tryptophanase structural gene (t
An approximately 2.3 kb fragment containing naA, ) was prepared.

Next, plasmid pBR322 DNA was digested with restriction enzyme BamH.
After complete decomposition by reaction at 37°C for 1 hour using I and Hindll, it was mixed with the approximately 2.3 kb fragment prepared earlier and heated at 65°C for 10 minutes to inactivate the restriction enzyme. After that, 50mM Tris buffer (pH 7)
, 6), 10mM dithiothreyl, 1mM AT
Add each component of P, 10 mM MgCI 2 and 1 unit of T4 DNA ligase (the concentration of each component is the final concentration), conjugate by reaction at 16 °C for 15 h,
Escherichia coli HB I O1 competent cells (manufactured by Takara Shuzo) were transformed using this solution. The obtained transformed strain was cultured in liquid by a conventional method, plasmid DNA was extracted from the culture solution, and restriction enzymes BamHI and H1ndI were added.
11, it was found that BamHI of pBR322
A DNA insertion of approximately 2.3 kb was observed at the HindlI site (hereinafter this plasmid is referred to as pBR322tnal).

(2) Preparation of m1ni-F fragment Escherichia coli strain 112 (ATCC15153
) with IQ medium (Bacto tryptone 10g,
5 g of yeast extract, 15 g of NaC, 1 g of glucose-7, water lα; p+-r7.2) was inoculated, and the
After shaking culture at 0 for about 4 hours, the bacterial cells were collected, treated with lysotium, and lysed by adding sodium dodecyl sulfate (SDS). This lysate was centrifuged at 32,000Xz for 40 minutes, the supernatant was fractionated, and then subjected to cesium chloride-ethidium bromide equilibrium density gradient centrifugation, followed by dialysis to obtain a solution containing the F factor plasmid. This fractionated solution was subjected to ethanol precipitation, and approximately 20 μ9 of F factor plasmid was finally collected.

Next, when preparing the mini-F fragment, 5 μ9 of the above F factor plasmid was taken, and the F factor plasmid DNA was treated with the restriction enzyme EcoRI at 3 μO for 1 hour.
D containing the approximately 9.2 kb miniF fragment by cutting the A chain
A NA solution was prepared.

(3) Construction of target plasmid Plasmid pBR322tnal obtained in the above (1)
The DNA was incubated at 37°C using the restriction enzyme EcoRT.
After reacting for 1 hour for complete decomposition, it was mixed with the mini-F fragment obtained in section (2) above, and heated at 65°C for 10 minutes to inactivate the restriction enzyme. Each component in 1 was added to 50mM Tris buffer pH as a final concentration.
7.6, l O m M M g C I 2, 10m
M dithiothreitol, 1. [mMATP and T4 ligase] Each component was added to form a unit, kept at 16°C for 15 hours, and ligated (hereinafter, this plasmid was referred to as pB
(May be tentatively named R 3 2 2 F tna)
.

Using the plasmid DNA prepared in section (3) above, Escherichia coli K12 strain (ATCC27325) was transformed according to a conventional method to obtain a transformed strain. This strain was cultured in liquid according to a conventional method, and plasmid I) B was extracted from the culture solution.
R 3 2 2 F tna was separated and purified. This plasmid was named pMTP4.

This plasmid pMTP4 was digested with various restriction enzymes,
The number of recognition sites and the size of degraded fragments were determined. The results are shown in Table 3.

9 0 Table 3 EcoRI 2 6.1 (9,2
), 4.2(6,3)H1ndlII 2
9.8 (14,7), 0.5 (0,8) ()
The numbers inside indicate kb.

The restriction enzyme cleavage point map of the plasmid pMTP4 described above is shown in FIG.

The transformed strain Escherichia coli YK3007 carrying this plasmid pMTP, 11 is the Ibaraki strain Tsukuba Southeast 1.
The contract number: FERM BP-2 was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, No. 1-3 Chome.
803).

(5) Stability of transformed strain Escherichia coli YK3007 The above-mentioned medium 10 containing 2 g/Q of glucose as a carbon source
Dispense 0 mQ into a 500 m + 2 volume Erlenmeyer flask,
The transformed strain obtained in Example 1 was inoculated into a strain that had been sterilized at °C for 15 minutes, and cultured with shaking at 37 °C for 24 hours.
The mixture was dispensed into a 12-volume Erlenmeyer flask, sterilized at 120°C for 15 minutes, and then subcultured at a rate of 50 calls per mQ, followed by shaking culture at 37°C for 24 hours.

Next, the bacteria were collected using centrifugation, and after washing the cells, a certain amount of ampicillin was added to the medium at a rate of 50 μg/mQ, and a fixed amount was spread on a plate medium prepared as an additive-free medium.
After culturing for 1 day at 7°C, grow colonies are counted.

As a result, it was confirmed that the number of colonies grown on ampicillin-added and non-ampicillin-added medium was the same, and that all L medium-grown colonies grew on tryptophan-free minimal medium (containing indole 10 mg/Q). The stability of was confirmed.

Transformation of strain 1 2 (1) Tryptophan synthase structural gene (trpA
, trpB) (A) Preparation of phage-80pt
) was inoculated into LOOMFF's medium (composition is the same as above) and shaken at 37°C for about 4 hours, and 0.2 m12 of the culture;
A phage d80 (ATCCI 1456a-Bl) aqueous solution (105 cells/m(1) of 0° lmff is mixed in L medium soft agar (L medium ten agar) and then layered on an L medium agar plate. When the plate was incubated at 37°C for about 5 hours, plaques (lytic plaques) were formed and
When the culture is continued at 37°C for ~3 days, a growing colony of 7 Fujime 80 lysogens is generated in the plaque. After culturing the lysogenic bacteria in L medium at 37°C for 4 hours, the same L medium as above was added.
After spreading on a medium agar plate, UV irradiation (40
Phage+ (80pt (phage DNA containing t-Lyptophan Oberon) is prepared by induction of lysogenic phage with 0-800ergs/mm2.10-20 seconds).

(B) Preparation of tryptophan operon fraction Escherichia coli strain 112 (ATCC 27325) was inoculated into 112 medium (composition is the same as above), cultured with shaking at about 37°C for about 3 hours, and reached the logarithmic growth phase. 10 m of 25% (w/v) glucose solution (2 and phage l8 prepared above)
Add 0 pL solution at a concentration of 1011 molecules/mQ moi
20) After continuing shaking for 5 hours, 80 pt of 7Argy was prepared in large quantities by adding chloroform in the usual manner [T
, Maniatis, E.

F, Fritschs S ambrook;
“Mo1ecular cloning (198
2) p, 76-80 Cold SpringHa
see rbor laboratory].

Next, 80 pL of the obtained phage solution was dialyzed against 1-Lis buffer (pH 7, 8), and then DN
Extraction method A [above “Molecular cloning”
p. 85] to extract and purify the phage DNA,
Restriction enzyme BamHI was added to this and reacted at 30°C for 30 minutes to obtain a tryptophan operon gene fraction with a length of about 20 kb.

(C) Preparation of plasmid pBR322 3 4 The tryptophan operon fraction obtained in section (B) above was reacted with restriction enzymes 5alI and XhoI at 37°C for 1 hour to obtain a DNA fraction with a length of approximately 7.4 kb. After cutting out, the reaction solution was heat-treated at 65°C for 5 minutes to inactivate the restriction enzyme, and then plasmid pBR322, which had been similarly treated with 5all restriction enzymes, was added and mixed.

The components in the quenching solution were then adjusted to a final concentration of 50% each.
m ML Lys buffer (pH 7,6), 10mM g
CI 2.10mM dithiothreitol, 1mM A
Each component was strengthened to 1 unit of TP, T, and DNA ligase, and the DNA was bonded by incubating at 16°C for 15 hours.

The recombined DNA was used to infect E. coli.
strain (tryptophan-requiring mutant, ATcc23718
) was transformed according to a conventional method to obtain a transformed strain [loss of Trp auxotrophy, i.e. LrpA, L
The rpB gene enables the biosynthesis of tryptophan, and minimal medium (K2HP0°7g, KH2PO421
J, Mg5O, 7H200゜1g, (NH=)2S
○, 1g of glucose, 2g of glucose, and a strain capable of growing on pure water (IQ)] was obtained.

This strain was cultured in liquid according to a conventional method, and a plasmid was isolated and purified from the culture solution (hereinafter, this plasmid was referred to as pB R3
22trp).

(D) Tryptophan synthase structural gene (trl)
Cloning of A% trpB) fraction Plasmid pBR322trp obtained in section (C) above was digested with restriction enzyme 5ac.
Cut with Il and 5alI at 37°C for 1 hour, and trpA
Both portions (approximately 3.5 kb in length) containing the trpB gene were obtained. Then, it was partially digested at 37°C using the restriction enzyme HindTI to obtain a minimal fraction (about 2.6 kb in length) containing trpAlLrpB. This fragment was mixed with a 5alI linker and ligated with T4 DNA ligase to obtain trpA and trpB fractions having 5alI sites at both ends.

(2) DNA containing the trypto-7anase promoter and the regulatory gene tnaC located downstream of the promoter
Preparation of fragments (A) Escherichia coli strain 112 (ATCC27
325) Preparation of chromosomal DNA 5 6 L medium (tryptone log, yeast extract 5 g, glucose 1 g, NaC 15 g, distilled water lQ; p87.2) l
O0ml was dispensed into an Erlenmeyer flask with a capacity of 500ml and sterilized at 120°C for 15 minutes. In this medium, E. coli (E5cherichia coli) was added.
) Inoculated with K-12 strain (ATCC27325),
After culturing at 37°O''c' for 15 hours, 2mα of this culture solution was taken, and 100mo of the above medium was added.

and cultured again at 37°C for 4 hours.

After culturing, the entire volume of the culture solution was centrifuged (8°000R).
PM, 15 minutes, 4°C) to collect bacteria, and dilute the bacterial cells to 50mM.
Tris buffer (pH 8,0)-10mM EDTA・
It was suspended in 2Na solution 5Qm12.

Next, lysotium was added to a final concentration of 2 mg/mQ, and after standing for 5 minutes, 6 ml (l) of 10% sodium dodecyl sulfate solution was added and kept at 65 °C for 30 minutes to lyse the bacteria. Add 15 mQ of 5M NaCl solution to the solution.
was added, cooled at 0°C for 1 hour, and the entire volume was centrifuged (12
00Orpm, 60 minutes, 40C), separate the supernatant fraction, add twice the amount of ethanol, mix, and centrifuge (
5000 rpm, 10 minutes, 4°C). The obtained precipitate was added to] 0mM Tris buffer (pl-17,5)
The DNA was dissolved in a -1mM EDTA/2Na solution, treated with phenol (protein removal treatment) and treated with RNA degrading enzyme, and finally 1.5 mg of DNA was obtained.

(B) DNA containing a tryptophanase promoter and the regulatory gene tnaC located downstream of the promoter
Preparation of fragments 25 μ9 of the chromosomal DNA prepared in the above (A) was treated with the restriction enzymes Hindn and BamHI (5 units each).
) for 1 hour at 30°O, and the chromosomal DNA
A HindnI and BamHI decomposition product solution was prepared.

To this digest solution, 1 μg of plasmid pBR3 was added with restriction enzymes HindIII and BamHI (1 unit each).
Mix the decomposition product solution obtained by reaction at 30°C for 1 hour using 50mM Tris buffer (pH 7°6).
), 10mM dithiothreitol, 1mM ATP,]
OmM MgC+ 2 and T4 DNA ligase 1 un
Each component of iL was added (the concentration of each component was the final 78 concentration) and allowed to react at 16°C for 15 hours to allow binding.

Using this solution, the conventional method [M, Mandel, A
.

Higa; J, Mo1. Biol,, 5
3.1.59 (1970)] according to Esc-herichia coli K
-12 strain (tryptophanase-deficient mutant and tryptophan auxotrophic mutant) was transformed, and selective medium [K2H
PO+71? , KH2POt2+? (NH4)
2so, Ig, MgSO4・7H,00,1g, casamino acid 5g, indole 10mg, adenine 2g, agar 2
0g, succinic acid 5Q+++9, pure water 1α].

The strain grown on this medium was inoculated into L medium containing anpinlin at a final concentration of 50 μg/mQ, and after culturing at 37°C for 7 hours, the bacterial cells were centrifuged (8000 PPM).

Collected at 4°C for 10 minutes. Tryptophanase activity was examined using this bacterial cell.

Reaction solution for tryptophanase activity measurement (0.1M phosphate buffer pH 8.0, 10mM tryptophan, 0.1%
Triton X-1X-100) 1 with wet bacterial cells 5011117
was added and reacted at 37°C for 30 minutes.

When the produced indole was colored using p-dimethylbenzaldehyde, a red colored strain was obtained. From this strain, the alkaline-3DS method [T, Mania
tis, E. F., Frltsch, J.
Sambrook;
ning (1982) p90-91 reference 1, and the plasmid was treated with the restriction enzyme BamHI.
and Hindn[, and the molecular weight was examined using agarose gel.
An approximately 3.2 kb insertion was observed in the dIff and BamHI sites (hereinafter this plasmid will be referred to as pB R322tna).
).

The obtained pB R322tna was digested with restriction enzyme A and luI.
and RsaT for 1 hour at 37°C to obtain two fragments approximately 630 bp in length containing the Trizoi fanase promoter and the regulatory gene tnaC. Furthermore, this fragment contains Ba
The mHI linker was mixed and ligated with T4 DNA ligase to obtain a tryptophanase promoter and regulatory gene LnaC fraction having BamH1 sites at both ends.

9 0 (3) Creation of target plasmid First, the DNA fractions obtained in the above (1) and (2) were each added to 5al of plasmid puc1.19 (manufactured by Takara Shuzo).
It was inserted into the T site and BamHI site according to a conventional method. Next, this plasmid was digested with restriction enzyme Ec.

Partial cleavage was performed with RI and HindlI to extract the tryptophanase promoter and regulatory gene tnaC and tryptophan synthase structural genes (trpB, t,
A DNA fraction containing rpA) was excised. This DNA fraction was used as Ec of plasmid pBR322.

It was inserted into the RT and HindllI sites according to a conventional method. Furthermore, the m1ni-F fraction prepared in Section (2) of Example 1 was inserted into the EeoRI site of this plasmid according to a conventional method to produce the desired plasmid.

(4) Transformation of Escherichia coli strain 112 with plasmid pMTY20
from the strain (ATCC27325) using the conventional method (Molecular Biology Experiment Manual, P supervised by Masaya Yogami.

A tributophanaze-deficient mutant strain was collected according to the following method (Kodansha, 1984), and transformed using the plasmid DNA prepared in section (3) above as a host to obtain a transformed strain. This strain was cultured in liquid according to a conventional method, and a plasmid was isolated and purified from the culture solution, and this plasmid was named pMTY20.

This plasmid pMTY20 was digested with various restriction enzymes, and the number of recognition sites and the size of the digested fragments were measured. The results are shown in Table 4.

Table 4 EcoR125,5(9,2),4.4(7,4)H1
ndI [35,6 (9,3), 3.0 (5,0), 1.
3(2,3) () indicates kb.

The restriction enzyme cleavage point map of the above-mentioned Nibras Middle MTY 20 is shown in FIG.

The transformed strain Escherichia coli YK2017 carrying this plasmid pMTY20 was sent to the Institute of Microbial Technology, Agency of Industrial Science and Technology, 1-1-3 Tsukuba Denshu, Ibaraki, with the accession number and Fiber Engineering Research Institute. No. 2804 (FERM
BP-2804).

(5) Stability of transformed strain Escherichia coli YK2017 The above-mentioned medium (carbon source, containing 2 g/Q of succinic acid instead of 1 g/ff of glucose) room (+500 m+2
The transformed strain Escherichia coli YK2017 obtained in section (4) above was inoculated into a volume Erlenmeyer flask and sterilized at 120°C for 15 minutes, followed by shaking culture at 37°C for 24 hours. After that, 100% of the medium prepared in the same way
Dispense +n12 into a 500mQ Erlenmeyer flask and incline at 120°.
50 cal per lmff sterilized at C for 15 minutes
The cells were subcultured at a ratio of ls, and cultured with shaking at 37°C for 24 hours.

Next, the cells were collected using centrifugation, and after washing the cells, a certain amount was spread on a plate medium prepared as L medium supplemented with ampinrin at a rate of 50 μg/mα and L medium without additives.
After culturing for 1 day at 7°C, growing colonies were counted.

As a result, it was found that the number of colonies grown on ampicillin-added and non-ampicillin-added media was the same, and that all colonies grown on L medium grew on a minimal medium (containing indole 10 mg/Q) that did not contain tryptophan. A high degree of stability of the plasmid was confirmed.

Transformation of strain (1) Preparation of plasmid pcAT-1 The DNA fragment (approximately 0.67 kb) containing the tryptophanase promoter with BamH1 sites at both ends and tnaC located downstream thereof, obtained in Example 2 (2). ) is a chloramphenicol acetyltransferase structural gene (approximately 0.66 kb) that does not have its own promoter.
Plasmid pKK232-8 (purchased from Pharmacia Strains) carrying the plasmid pKK232 was inserted into the Bam3 4 HI site according to the standard method.
-8CAT was created. Furthermore, pKK232-8CA
m1 obtained in Example 1 (2) at the S ma I site of T
Insert the ni-F DNA fragment (approximately 2 kb) to create the target plasmid pCA"r-1.

The sizes of each fragment when plasmid pCAT-1 is cut with various restriction enzymes are shown in Table 5 and in Figure 3.
c: Restriction enzyme cleavage point map of AT-1 is shown.

Cloth-) EcoRI 3 6.6 (10,0)
, 3.1 (4,7), 0.07 (0,1) BamHT
3 7J7 (115), 1.7 (2,
6), 0.4(0,7) Sal I2
6.47(9,8), 3.3(5,0) () indicates kb.

(2) Transformation of Escherichia coli strain-112 with plasmid pCAT-1 Next, transformation of Escherichia coli strain-12 (ATCC27
325) was transformed as a host using the plasmid pCAT-I DNA prepared in section (1) above according to a conventional method. The obtained transformed strain was transformed into Escherichia coli CA
It was named T-1.

(3) Stability of transformant Escherichia coli CAT-1 100 mQ of the above medium (containing 2y/Q succinic acid instead of 1 g/Q carbon source Nigelcose) was dispensed into a 500 m4 Erlenmeyer flask and heated to 120°C. The transformed strain E 5cher obtained in section (2) above was sterilized for 15 minutes.
Inoculated with ichia coli CA T-1, 37
After 24 hours of shaking culture at °C, 1 m72 of the similarly prepared medium Loom (2) was dispensed into 500 m (2 volume Erlenmeyer flasks and sterilized at 120 °C for 15 minutes).
The cells were subcultured at a ratio of 50 calls per cell, and cultured with shaking at 37°C for 24 hours.

Next, the bacteria were collected using centrifugation, and after washing the bacterial cells, a certain amount was spread on plate cultures prepared as L medium supplemented with ampicillin at a rate of 50 μg/mfl and non-additive medium. After culturing for 1 day at °C, growing colonies were counted.

As a result, it was found that the number of colonies grown in ampicillin-added and non-ampicillin-added media was the same, and that all L-medium-grown colonies grew in L-medium supplemented with chloramphenicol at a rate of 50 μg/mQ. High stability was confirmed.

Dispense 100 mff of the medium shown in Table 6 into a 500 m 12 Erlenmeyer flask, sterilize it at 120°C for 15 minutes, and add a 50% (WT/VOL) glucose solution (120°C
! After aseptically adding 1 m+2 of (sterilized for 115 minutes), Escherichia coli (E 5cherichia coli) YK3, the tryptophanase producing bacteria obtained in Example 1
007 (FERM BP-2803) and 3
After culturing with shaking at 7°C for 1 day, 20 mQ of the culture was treated in the same manner, except that glucose was at the upper limit concentration of each test group in Table 6 (
Inoculate 100,100 O of the prepared medium at 37°C with a rotation speed of 60 ORPM and aeration volume of 1 V at 37°C using an aerated stirring culture tank so that
vm, pH 7.2 (adjusted with 28% ammonia water) for 8 hours. Note that glucose is 50% (WT/
VOL) (7) While measuring the glucose concentration in the culture solution over time using an aqueous solution (sterilized at 120°C for 15 minutes), make sure that the concentration does not exceed the upper limit for each experimental group shown in Table 7. , and the glucos concentration was 0.01% (wt/v
The addition was carried out continuously in an aseptic manner so that the amount did not go below 100 ml, and the addition was stopped when the total amount added in each experimental group reached 209 ml.

Table 6 L-) Libutophane H2PO4 with 2HPO4 (NH1)2 S Ot MgSO, 7H20 Fe SOa 7 H2O Casamino acid (DTFC○) Add distilled water (pH 7°8 2) 0.5g 1.6g 5 , 5g 3, 0g O, ig 0mg 5, 0g 000mQ After completion of the culture, 20 m4 of each culture solution was centrifuged and tryptophanase activity was measured using the method described above. The results are shown in Table-7. In addition, as a comparative example, a bacterial cell cultured only by adding 20 g/ρ of glucose at the initial stage of culture was used.

Sac-1- * Comparative example (glucose 20g/Q added at the start of culture)
Relative value with the activity of
After aseptically adding 1 m+2 of 50% (WT/VOL) glucose solution (sterilized at 120°C for 15 minutes) to the 2-volume Erlenmeyer flask and sterilizing it at 120°C for 15 minutes, the mixture obtained in Example 2 was added. Escherichia coli, a tryptophan synthase-producing bacterium,
li) YK 2017 (F E RM BP-28
04) was inoculated and cultured at 37°C for 10 times with shaking, 20 mQ of the culture was prepared in the same manner, except that glucose was adjusted to the upper limit concentration (wt/vo 1%) of each test group in Table 8. Inoculate the medium 1000n+12 and use an aerated stirring culture tank at 37°C, rotation speed 60 ORPM, aeration volume 1 vv.
The cells were cultured for 8 hours at pH 7.2 (adjusted with 28% aqueous ammonia). Note that glucose is 50% (WT/V
While measuring the glucose concentration in the culture solution over time using an aqueous solution (sterilized at 120°C for 15 minutes) of OL), the concentration upper limit (WT/VOL) for each experimental group shown in Table 7 was measured.
%) and 0. O1% (WT/VOL)
The glucose was added continuously in an aseptic manner so as not to fall below the lower limit, and the addition was terminated when the total amount of glucose added to each experimental group reached 20 g.

90 After completion of the culture, 20 ml of each culture solution was centrifuged, and tryptophan synthase activity was measured using the method described above. The results are shown in Table 8. In addition, as a comparative example, a bacterial cell cultured only by adding 20 g/Q of glucose at the initial stage of culture was used.

L Yu* Comparative example (glucose 20g/Q added at the start of culture)
100 mD of the medium shown in Table 6 of Example 4 was taken as 500 m0
．． After dispensing into Erlenmeyer flasks and sterilizing them at 120°C for 15 minutes, 1 m+2 of 50% (WT/vot) glucose solution (sterilized at 120°C for 115 minutes) was added aseptically, and the product obtained in Example 3 was obtained. Escherichia coli (E 5ch
Erichia coli) CAT-1 was inoculated,
After culturing with shaking at 37°C for 1 day, 20 mQ of the culture was added to 100 mQ of the culture medium prepared in the same manner, but with glucose at the upper limit concentration (wt/vo 1%) for each test group in Table 8.
Inoculated at 0 mff, using an aerated stirring culture tank at 37°C, rotation speed 600 rpm, aeration volume 1 vvm, pH
7.2 (adjusted with 28% ammonia water) for 8 hours. Glucose was determined using a 50% (yH/vol) aqueous solution (sterilized at 120°C for 15 minutes), and while measuring the glucose concentration in the culture solution over time, the concentration for each experimental group shown in Table 9 was determined. The addition was carried out continuously in an aseptic manner so that the upper limit was not exceeded and the glucose concentration did not fall below 0.01% (wt/vol), and the total amount added in each experimental group was 20 g. The culture was terminated at this point.

After completion of the culture, 20 mQ of each culture solution was centrifuged, and chloramphenicol 12 acetyltransferase activity was measured using the method described above.

The results are shown in Table 9. In addition, as a comparative example, glucose 20g10. Bacterial cells cultured only by adding .

Consideration* Comparative example (glucose 20g/Q added at the start of culture)
Production of relative value with activity of 1 as shown in Table 6 of Example 4.
In Example 2, a 50% (WT/VOL) glucose solution (sterilized at 120°C for 115 minutes) was added to a 2-volume Erlenmeyer flask and sterilized at 120°C for 15 minutes. The obtained Toriji 1-Huan synthase producing bacteria, Escherichia coli (Escherichia coli)
coli) YK-2017 (FE RMBP-2
804) and cultured with shaking at 37°C for 1 day, 20 m<1 of the culture was inoculated with 10100 O of a medium prepared in the same manner.
, using an aerated stirring culture tank at 37°C, rotation speed 600 rpm, and aeration volume 1 vvm.

Culture was carried out at pH 7.2 (adjusted with 28% ammonia water). Note that glucose was determined using a 50% (WT/V○L) aqueous solution (sterilized at 120°C for 15 minutes) and the glucose concentration in the culture solution was measured over time as shown in Table 2. The addition was carried out continuously in an aseptic manner so as not to exceed the upper limit of the concentration in each experimental group, and the culture was terminated when the total amount added in each experimental group reached 20 g.

Further, the dissolved oxygen concentration in the culture solution was maintained at the value for each experimental group shown in Table 10 by aerating oxygen gas instead of air. In addition, in a system where oxygen is not aerated, the dissolved oxygen concentration reaches 0 after about 4 hours.
.

After completion of the culture, 20 mQ of each culture solution was centrifuged, and tryptophan synthase activity was measured using the method described above. In addition, as a comparative example, glucose 20
Bacterial cells cultured only by adding g/Q were used. The results are shown in Table IO.

Table 10 *1 Cultivation with 20g/Q of glucose added at the start of culture *2 Relative value when the activity and bacterial body recovery amount of the comparative example without oxygen concentration control is taken as 1 *3 Dissolved oxygen concentration was not controlled by oxygen aeration things

[Brief explanation of drawings]

FIG. 1 shows a restriction enzyme breakpoint map of plasmid pMTP4. FIG. 2 shows a restriction enzyme breakpoint map of plasmid pMTY20. FIG. 3 shows a restriction enzyme breakpoint map of plasmid pCAT-1. 5 6

Claims (1)

[Claims] 1. A DNA fragment (a) containing the promoter in the tryptophanase operon (tna) and the regulatory gene tnaC located downstream of the promoter, and a DNA fragment (b) containing the target structural gene that can be expressed by the promoter.
) and glucose as a carbon source at a concentration of 0.01 to 0.3% (wt/vol. ), the transformed microorganism is cultured while being continuously or intermittently added to the medium so as to maintain the above-mentioned target structural gene in the microorganism, thereby causing the microorganism to express the target structural gene. Method.