JPH0665307B2 - Bacillus subtilis operon gluconate and promoter - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to Bacillus subtilis.
Gluconate operon and promoters that control its transcription.

The present invention particularly relates to a DNA containing a portion that inducibly enables transcription of an operon by adding gluconic acid into a medium.
Regarding the base sequence.

With the recent development of genetic engineering, it has become possible to produce a protein encoded by the gene in a host microorganism by introducing a recombinant DNA in which a heterologous gene is linked to a vector into a microbial cell. When attempting to produce a substance using such a technique, Bacillus bacteria have been used in the fermentation industry for a long time, and since they are not pathogenic and do not produce pyrogens,
It is well known that it is more suitable as a host microorganism than Escherichia coli which is widely used in genetic engineering research. However, when a heterologous gene is expressed in a bacterium belonging to the genus Bacillus, RNA polymerase and ribosomes of the bacterium belonging to the genus Bacillus have a strict specificity with respect to recognition of a promoter region and a ribosome binding region (Sueji Horinouchi, Protein Nucleic Acid Enzyme 28 1468 ( 1
983)) It is necessary that those regions originate from the genus Bacillus (Goldfarb, DSet.
al.) Nature 293 309 (1981)). From this point of view, a promoter derived from the genus Bacillus, which enables the expression of a heterologous gene in a bacterium belonging to the genus Bacillus, is an important key for producing substances by genetic engineering. In addition, the following two points have been conventionally problematic with respect to the timing of expression of a heterologous gene from another viewpoint of substance production by genetic engineering.

The first problem is that in the case of producing a substance that is harmful to the host microbial cells, if the gene encoding the substance is expressed in the early stage of the growth of the host microorganism and the substance is produced, the host microorganism is At that point, it will die and the production of the substance will be impossible. In order to avoid such a situation, the expression of the gene may be started after the growth of the host microorganism is completed. That is, expression of the gene may be induced after the growth of the host microorganism is completed by some means. However, there are only a few known examples of promoters involved in inducible expression in Bacillus bacteria, which are conventionally induced by antibiotics (Hardy (K. Hard
y) et al. Nature 293 481 (1981)), Williams (DMWilliams) et al. Gene 16 199 (1981))
In these cases, there is a big problem in that an expensive antibiotic is used as the inducer and a step of finally removing the antibiotic from the target substance is required.

On the other hand, when the produced substance is harmless to the host microorganism, it is said that a large amount of the substance is accumulated in a short time when the gene encoding the substance is expressed from the early stage of growth and the substance is produced. From the viewpoint, the above problem does not occur.

The second problem is a phenomenon known as catabolite repression when there is a sugar source in the medium, but it is the case where the function of the promoter is suppressed and the gene expression is suppressed as a result. This point is also a major problem in terms of substance production, and in order to avoid this, it is necessary to perform long-term culture or remove the sugar source from the medium.

This requires extra utility costs or makes it impossible to use inexpensive molasses in the medium, and is an industrially early point in material production.

In view of such a point, the present inventor has repeatedly studied an inducible enzyme of the genus Bacillus that can be induced at an arbitrary culture time by the addition of an inexpensive substance and expression does not occur before the induction, and finally, easily from glucose. It was found that the inexpensive gluconate operon involved in gluconate assimilation is rapidly induced by the addition of gluconate, and the promoter controlling the expression of the operon and the operon linked thereto are isolated as a DNA fragment. The separation was successful, the base sequence was determined, and the present invention was completed.

Furthermore, as a result of examination using the promoter search plasmid pPL603B, when a base sequence near the promoter of the DNA fragment was inserted into the plasmid,
Confirming that the expression of the chloramphenicol resistance gene of pPL603B may be expressed by gluconic acid or may be expressed without being suppressed even in the presence of glucose, confirming the practicality of the present invention did. That is, it was proved by the present invention that the above-mentioned two problems in the substance production using the genetic engineering method using Bacillus as a host can be solved at once.

The present invention will be described in detail below.

DN containing gluconate operon and its promoter
Any DNA donor strain for obtaining the A fragment may be used as long as it is Bacillus subtilis having gluconic acid assimilation. That said, the enzymes involved in the assimilation, namely, gluconate permease and gluconate kinase encoded in the gluconate operon, are strictly subjected to catabolite repression by glucose, and glucose is not present in the medium. It is preferable for the purpose of the present invention that it is strongly induced by gluconic acid under the conditions. Any Bacillus bacterium can be used as a cloning host for obtaining the DNA fragment, but Bacillus subtilis is preferable from the viewpoint of progress of genetic analysis, and a gluconic acid-assimilating deficient strain is more preferable because of easy selection. Is convenient.

In the present invention, the operon means a gene group which is one transcription unit linked to a promoter as in the usual concept. The promoter means a site at which RNA polymerase binds to start transcription of an operon. Therefore, the gluconate operon and promoter referred to in the present invention are one transcription unit containing genes encoding gluconate kinase and gluconate permease, which are enzymes required to assimilate gluconate, and mRNA synthesis of the transcription unit. For the RNA polymerase binding site. In order to obtain a DNA fragment containing such a gluconate operon and a promoter, the usual cloning technique of Bacillus subtilis may be used without any special technique. The technology is described in DADubnau, The molecular Biology of the Bacilli, Academic Press 1982, OLD, Primrose by RWold, SBPrimrose, Principles of Gene Manipulation. (Principles of Gene M
anipulation), Blackwell Scientific Publications (Blackwell Scientific Publicati
ons) 1981), the vector used for cloning may be either a phage or a plasmid. For example, as a phage vector, 911, φ10
5, φ3T and the like are often used, and pUB110, pC194, pE194, pBD64 and the like are often used as the plasmid vector, and any of them can be used in the practice of the present invention. When Bacillus tin butyris deficient in gluconate utilization is used as a cloning host, it becomes easy to obtain the target DNA fragment containing the gluconate operon and its promoter. That is, it is only necessary to select a transformant strain that has acquired the ability to utilize gluconate.

Recombinant DNA from the transformant thus obtained
No special method is required to obtain normal phage DN
A, a method for preparing plasmid DNA may be used. Regarding these methods, a book written by T.maniatis et al. (Molecular Cloning),
Cold Spring Harbor Laboratory (Cold Spring
Harbor Laboratory) (1982)). The obtained recombinant DNA can be digested with a restriction endonuclease and then subjected to agarose or polyacrylamide gel electrophoresis to determine the cleavage site to prepare a cleavage map. Obtained recombinant DNA
The nucleotide sequence of the DNA fragment inserted into the vector can be determined by any of the commonly used methods. As such a method, Maxam-Gilbert method (A.Maxam, W.Gilbert), Procedure Dings National Academy of Sciences USA (Proc.Natl.
Acad.Sci.USA) 74 560 (1977)) or a method using phage M13 (J. Messing et al., Nucl. Acids Res.), 9 309 (1).
981), Tsagursky, Bellman (RJZagursky, MLB
erman) and Gene 27 183 (1984)) are well known.

The nucleotide sequence of the DNA fragment containing the gluconate operon and the promoter thereof obtained in the present invention is as shown in claim 2, and FIG. 1 schematically shows this sequence. In this sequence, the −35 region and −10 region, which are the base sequences for recognition and binding of RNA polymerase found in the promoter, are common to the promoters recognized by Sigma 55 RNA polymerase in TTGCAT and TATCAT, respectively, in the present invention. Array
It is a type similar to TTGACA and TATAAT that allows efficient transcription.

It is also known that Bacillus subtilis (b. Subtilis) does not allow the expression of heterologous genes because of the problem of ribosome-mRNA binding in the process of translation from mRNA to protein. Of the Biological Chemistry (J.Biol.Chem.) 256
11283 (1981)) suggests that strong binding between the nucleotide sequence downstream of the promoter known as the ribosome binding region and the 3'end of 16S ribosomal RNA is important for expression. Nucleotide sequence of the first ribosome binding region of the protein that is present downstream of the promoter of the gluconate operon of the present invention is AGGAAGG, and with 3 'terminal 3' _OH UCUUUCC ⁵ 'good complementarity ribosomal RNA of Bacillus subtilis There is. Therefore, it is agreed that the ribosome binding region of the gluconate operon of the present invention is of a type capable of strong expression in Bacillus subtilis.

This means that when a DNA fragment containing a promoter portion of the present invention is inserted into a promoter search vector, the chloramphenicol resistance gene (chloramphenicol acetyltransferase gene) is expressed and 100 μg / ml of the antibiotic substance in a synthetic medium. It is also obvious to give resistance to. And in that case, when a short DNA fragment containing a promoter is inserted, chloramphenicol acetyltransferase is expressed in the host Bacillus subtilis at a high level in the presence of glucose without catabolite repression and at the same level as in induction.
Give 0 μg / ml of the antibiotic resistance. When a long DNA fragment containing a promoter is inserted, expression of chloramphenicol acetyltransferase is normally suppressed, but it can be induced by adding gluconic acid to the medium, and 100 μg / ml of the antibiotic is added to the host Bacillus subtilis. It conferred resistance and this induction was shown to undergo cataporite repression by glucose.

What has been stated above is that by using the nucleotide sequence of the present invention, it is possible to express a heterologous gene constitutively without being subjected to catabolite repression depending on conditions, or to induce it by adding gluconic acid at any time. It also shows that it can be expressed in Escherichia coli, and has great significance in the substance production using genetic engineering.

The base sequence of the present invention is also a base sequence in the case where any part of the base is replaced with another base, a new base is inserted, or a base sequence is deleted in accordance with common knowledge of molecular biology regarding genes. In some cases, the derivative obtained when a part of is transferred has the effects of the present invention described above,
Such a thing naturally falls within the scope of the present invention.

The present invention will be described below with reference to specific examples, but the present invention is not limited to these examples.

Example 1 (Cloning of Bacillus subtilis gluconate operon) Bacillus subtilis M168 (trpC2) strain (Ohio Bacillus Stock Center stock: stock number 1A1)
After culturing at 37 ° C. for 15 hours in a broth medium (Nutrient Broth, manufactured by Difco), cells were collected and the method of Saito-Miura (Biocica Biophysica Acta (Bioc
him.Biophys.Acta) 72 619 (1963)) 5 mg of purified D
I got NA. The purified DNA was further purified by cesium chloride density gradient centrifugation, and 5 μg of the purified DNA was completely digested with 10 units of restriction nuclease EcoRI (Takara Shuzo) at 37 ° C. for 6 hours. The composition of this reaction system is 100 mM Tris-HCl (pH 7.5), 7 mM MgCl ₂ , 50 mM NaCl, 7 mM
2-mercaptoethanol, 0.01% bovine serum albumin.

The reaction product was purified by phenol extraction and ethanol precipitation, and on the other hand, the resulting product was subjected to the resulting reaction with phage φ105 DNA (5 μg) which had been similarly digested with EcoRI and T ₄ ligase (Takara Shuzo). T for this binding reaction
10 units of ₄ ligase were used. The composition of this reaction system is 66
mM Tris-HCl (pH 7.6), 6.6 mM MgCl ₂ , 10 mM dithiothreitol, 1 mM ATP. The reaction was carried out at 15 ° C for 4 hours. The recombinant phage thus obtained was then used to transform Bacillus subtilis. As a host bacterium, a mutant strain (Gn
t ^-) and is Bacillus subtilis 61,656 shares (FERMP-7834)
Was used. Prior to transformation with the recombinant phage, 61656 strain was first infected with phage φ105 to obtain 61656 strain in which the phage was lysogenized. Competent cells of this lysogen were prepared according to the method of Saito et al. (H. Saito et al., Journal of Genetics Applied Microbiology (J.Gen.Appl.Microbiol.) 7 243 (1961)). Transformation was carried out using the recombinant phage DNA described above. The details of this method were introduced by Kawamura (Fujio Kawamura Protein Nucleic Acid Enzyme)
26 469 (1981)).

As a result of this transformation, 250 strains of gluconic acid-assimilating strain (Gnt
⁺ ) Was obtained. 61656GP, one of the transformants
From the strain (FERM P-7835), a phage was induced by mitomycin C, and a 61656 strain in which the phage was newly lysogenized was constructed using the phage, and all were gluconate-utilizing (Gnt ⁺ ). . The strain [61656 (φ 105 Gn
The activities of gluconate kinase and gluconate permease of (t ⁺ )] are shown in Table 1. The activity of gluconate kinase and gluconate permease is determined by the method of Bergmeier et al. (HUBergmeyer (HUBergmeyer
et.al.) Method of Enticmatic Analysis (Me
thods of Enzymatic Analysis) Page 457 Academic Press (1974)) and the method of Dows et al. (Biochim.
s.Acta) 541 18 (1978)).

From this table, it is clear that gluconate kinase and gluconate permease activity can be induced in the transformed strain.

From this, it is clear that the structural genes for gluconate kinase and gluconate permease and their expression-regulating genes, that is, the gluconate operon and its promoter, have been cloned into φ 105 Gnt ⁺ obtained here.

Example 2 (Creation of cleavage map by restriction endonuclease and determination of nucleotide sequence) From 61656GP (φ105Gnt ⁺ ) obtained in Example 1 to φ105Gnt
⁺ Phage DNA was prepared, cleaved with restriction endonuclease EcoRI according to a conventional method, and its cleavage pattern was examined by agarose gel electrophoresis.

As a control, φ105 phage DNA similarly digested with EcoRI was used. As a result, it was revealed that the gluconate operon and its promoter were cloned in an EcoRI fragment of about 7 Kb, and therefore a restriction endonuclease cleavage map was created. The cut map is shown in FIG. Furthermore, it was revealed that the gluconate operonto and its promoter are present in the approximately 4.5 Kb DNA fragment of HindIII shown in this figure. Therefore, according to the method of Maxam-Gilbert, D
The base sequence of the NA fragment was determined. The results are shown in the claims. FIG. 2 schematically shows this result. As can be seen from this figure, there are four open reading frames encoding a promoter and a protein in this nucleotide sequence. From this, it was revealed that the gluconate operon encodes two proteins for regulation in addition to gluconate kinase and gluconate permease.

Example 3 (D near the promoter of gluconate operon
Examination of Expression of Heterologous Gene by NA Fragment) Promoter Search Vector pPL603B (Cloning Site is Bam) for Inserting a DNA Fragment containing a Promoter to Express a Chloramphenicol Resistance Gene (Chloramphenicol Acetyltransferase Gene)
HI) was used to investigate the expression of heterologous genes. That linked by T ₄ ligase by inserting a conventional method Sau3AIDNA fragment obtained from HindIII4.5Kb fragment into the cloning site of pPL603B containing restriction endonuclease gluconate operon and the promoter was partially digested with Sau3AI. Bacillus subtilis 1A using the recombinant plasmid
423 shares (Ohio University Bacillus Stock Center Preserved Strains) in Jiang's way (S.Chang, SN
Cohen) Molecular General Genetics (Mo
1) Gen. Genet.) 168 111 (1978)). One of the transformants obtained as a result, 1S21 (FERMP-
7832) contains an inserted DNA fragment of about 1.5 Kb and gives chloramphenicol resistance of 100 μg / ml when induced by gluconic acid, but this induction does not occur in the presence of 10 mM glucose and is regulated by catabolite repression. It became clear. Transformed strain obtained separately
1S22 (FERM P-7833) contains an insert DNA fragment of about 0.4 Kb, which is 100 μg / ml even in the presence of 100 mM glucose.
It was clarified that this confers resistance to chloramphenicol.

From this result, using the gluconate operon of the present invention and the promoter thereof, it is possible to arbitrarily express a heterologous gene according to the purpose, even inducibly and as a constitutive one that does not undergo catabolite repression. It is clear that The medium composition used for the expression of this chloramphenicol acetyltransferase is 100 mM 4-morpholinopropane potassium sulfate buffer (pH 7.0), 10 mM ammonium sulfate, 5 mM potassium phosphate buffer (pH 7.0), 1 mM MgCl ₂ , 0.7 mM CaCl ₂ , 50 μM MnCl
₂ , 1 μM ZnCl ₂ , 5 μM FeCl ₃ , 5 g / casamino acid (Difco), 50 μg / ml L-tryptophan, 50
μg / ml L-methionine and glucose were added at a concentration of 10 or 100 mM as needed. Gluconic acid, which is an inducer, was added at 10 mM. The culture was performed at 37 ° C.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a restriction endonuclease cleavage map of a DNA fragment containing the gluconate operon and its promoter. It contains the gluconate operon and its promoter in a HindIII fragment of approximately 4.5 kb. The base sequence was determined using this portion. FIG. 2 is a schematic diagram of the gluconate operon and its promoter. ATrich: Sequence in which adenine and thymine are frequently found -35: -35 region of promoter -10: -10 region of promoter SD: Ribosome binding region, respectively. Note that Frames A, B, C, and D represent open reading frames encoding proteins.

Claims (2)

[Claims] 1. A promoter which controls transcription of the gluconate operon of Bacillus subtilis and a DNA of about 4.5 Kb containing the gluconate operon, which is under the control and whose transcription is regulated by catabolite repression.
A DNA fragment obtained by ligating a heterologous gene immediately after a DNA fragment of about 1.5 Kb obtained by partially digesting the fragment with a restriction enzyme Sau3AI. 2. A promoter that controls transcription of the gluconate operon of Bacillus subtilis and a DNA of about 4.5 Kb containing the gluconate operon, which is under the control and whose transcription is regulated by catabolite repression.
The DNA fragment according to claim 1, wherein one strand of the fragment has a base sequence shown below.