JPH06121677A - Method for highly manifesting enzyme gene acylating macrolide antibiotic - Google Patents

Abstract

PURPOSE:To efficiently manifest activity of an enzyme acylating the 4''-position of an acylating macrolide antibiotic. CONSTITUTION:A microorganism having an enzyme gene capable of acylating the 4''-position of a macrolide antibiotic (tylosin, spiramycin, angolamycin or deltamycin) is transduced with DNA fragment containing acyB2 gene obtained from a bacterium belonging to the genus Streptomyces to amplify the enzyme gene capable of acylating 4'' of the macrolide antibiotic.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a DNA fragment encoding a protein having an action of enhancing the expression efficiency when an enzyme gene for acylating the 4 "-position of a macrolide antibiotic is introduced into a host and expressed. Regarding usage.

Macrolide antibiotics (14-membered ring and 1
The 6-membered ring is a useful antibiotic that is widely used as a medicine and a veterinary medicine (for domestic animals and fisheries). Acylation of the 4 "-hydroxyl group of 16-membered macrolide antibiotics increases antibacterial activity by S. Omura et al. [Journal of Antibiotics].
Antibiotics) 28, 401-433 (1980)] and R.A. Okamoto et al. [Journal of Antibiotics
of Antibiotics) Volume 27, 542-54
4 (1979)]. One of the 16-membered macrolide antibiotics, tylosin-producing bacterium Streptomyces fladiae (Streptomy)
ces fradiae) produces only unacylated at the 3 and 4 "positions, that is, the -OH form. In order to acylate this, tylosin is once separated and then chemically or chemically isolated. Recently, a method for directly producing a 4 "-acylated tylosin by a genetic engineering method has been disclosed (JP-A-2-728).
No. 80).

The present invention enhances the expression of the 4 "-position acylation product in the case of producing a 4" -position acylation product by using the macrolide antibiotic 4 "-position acylating enzyme gene, and highly efficiently Which encodes a protein capable of producing
A method of using the A fragment is provided.

[0004]

2. Description of the Related Art The following macrolide antibiotics have been known, for example. Tylosin,

[Chemical 2] Angoramycin,

[Chemical 3] Spiramycin,

[Chemical 4] Leucomycin,

[Chemical 5] Deltamycin,

[Chemical 6] As a genetically engineered method for acylating the 4 "-position of these macrolide antibiotics, Janet Kay Ep et al. (JP-A-2-13380) encodes 4" -O-isovaleryl acylase. The use of DNA fragments containing In this method, a DNA fragment encoding a 4 "-position acylating enzyme is inserted into a vector plasmid for the purpose of genetically acylating a macrolide antibiotic having an 4" -OH position, and the host is transformed with the plasmid. Then, the obtained transformant is cultured. However, according to this method, the expression of the gene was weak, and 4 "-O-
It was not sufficient for the industrial production of isovalerylated macrolides.

[0005]

DISCLOSURE OF THE INVENTION The present invention aims at enhancing the expression of a 4 "-position acylating enzyme gene in a microorganism having a macrolide antibiotic 4" -position acylating enzyme gene. It is to provide a method for efficiently producing a ride antibiotic.

[0006]

[Means for Solving the Problems] The inventors of the present invention have earnestly studied to industrially and economically produce a 4 "-position acylated macrolide by expressing a 4" -acylation enzyme gene of a macrolide antibiotic. As a result of repeated studies on the expression mechanism of the 4 "-position acylase gene, the inventors independently isolated acyB2 DNA fragment (Japanese Patent Laid-Open No. 7288/1990).
No. 0) is deeply involved in the expression of the 4 "-position acylase gene, and it was found that the acyB2 DNA fragment significantly promotes the expression, and the present invention has been completed.

Thus, according to the present invention, Streptomyces thermotolerance
A size of about 2.1 kilobases (kb) derived from the thermotolerans ATCC11416 strain and a DNA fragment specified by the nucleotide sequence shown in FIG. 1 is provided. The DNA fragment acyB2 or its DNA restriction fragment of the present invention is useful for industrially producing a 4 "-acylated tylosin using a 4" -acylase gene, and is a macrolide antibiotic of the genus Streptomyces. It is particularly useful in that it allows industrial application of recombinant DNA technology to a microorganism that produces the substance. Hereinafter, the production of an acylated macrolide antibiotic by a transformant using a DNA fragment containing the gene acyB2 of the present invention will be described in more detail.

DNA containing the gene acyB2 of the present invention
As a strain of the genus Streptomyces which is the origin of the fragment, any strain can be used as long as it has the ability to produce a 4 "-position acylating enzyme of a macrolide antibiotic. For example, Streptomyces・ Streptomyces kitasatoens
is), Streptomyces narvonensis baru
Josamy Thetics (Streptomyces n)
arbonensis var josamyceti
cus), Streptomyces hygroscopic
us), Streptomyces platensis (Stre
ptomyces platensis, Streptomyces
s albireticuli), Streptomyces c.
inerochromogenes), Streptomyces jakartensis (Streptomyces)
djakartensis), Streptomyces fur
dicdicus), Streptomyces macrosporus
reus), Streptomyces tendae (Stre
ptomyces tendae, Streptomyces thermotolerance (Streptomyces t)
hermotolerans), Streptomyces
Deltae (Streptomyces deltae)
Among them, Streptomyces thermotolerance is preferable.

<Preparation of acyB2 fragment> ac of the present invention
The yB2-containing fragment can be obtained from the known plasmid pAO207. In addition, this plasmid can be obtained from the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, Micro Engineering Research Center No. 1880 (F
ERM BP-1880) can be extracted from the deposited strain. The above strain was aerobically cultured at 28 ° C. to obtain the strain, and the method of Hopwood et al. [Genetic Manipulation of Streptomyces (Genetic Manipulati) was obtained.
on of Streptomyces: A Laboratory Manual (A Laboratory Manual)
al), John Innes Foundation (Joh)
n Inners Foundation, Norwich, UK, 1985], to prepare plasmid pAO207. Further, among the fragments inserted into pAO207, acyB1 and acyB shown in FIG. 3A.
The SphI-SacI fragment of about 3.4 kb containing 2 can be subcloned using an appropriate method well known in genetic engineering.

Examples of the vector used at this time include E. coli vector pUC18 and actinomycete vector pI.
Various materials such as J702 can be used, but especially Escherichia coli-actinomycete shuttle vector pUJ350 (JP-A-2
-72880) and pSAN-lac (Example 1) are convenient. The fragment subcloned in this way can be prepared in large quantities as a plasmid. Next, in order to obtain a fragment containing these plasmids acyB2, an appropriate restriction enzyme capable of excising the fragment may be selected. For example, from the restriction enzyme map of the inserted fragment as shown in FIG. As shown in PstI-Sp
By digesting with hI, an acyB2 fragment with a size of about 2.1 kb (kilobase) can be obtained as shown in FIG. 3B.

In this acyB2 fragment, the DNA sequence up to the 1630th position with the first base (cytosine) at the PstI site as 1 is shown in FIG. Furthermore, the amino acid sequence of the protein encoded from the 450th position to the 1610th position of this fragment is shown in FIG.

<Functional analysis of acyB2> In general, in order to investigate whether the expression of a gene in the same organism is regulated by other factors such as suppression or promotion, the factor having the regulatory function is functioned by some method. It seems that the most appropriate method is to investigate whether or not the expression is deregulated in the case where it is abolished. From this point of view, to explain that the high expression of the macrolide-4 ″ -acylating enzyme gene acyB1 is achieved by the protein encoded by acyB2, the expression of acyB1 in the case where the protein is made non-functional can be explained. This can be proved by looking at the decrease, and there are several possible means for disabling the protein encoded by acyB2, but there are three main ones.

First, Streptomyces thermotoler which is the origin of acyB1 and acyB2.
ans) is chemically mutated with NTG or the like to obtain a mutant strain in which acyB2 function is lost. Secondly, from a recombinant plasmid containing acyB1, for example, a plasmid such as pMAB8 described in Example 2, acyB1
Streptomyces lividans (Str) transformed with the plasmid obtained by deleting most of the B2 region
eptomyces lividans) TK24. Third, Streptomyces lividans (Streptomyces lividans) transformed by frameshift mutation in the protein coding region of acyB2 in the above-mentioned pMAB8 recombinant plasmid was transformed.
Dans) TK24. Therefore, these strains are suitable for studying the action of acyB2 on acyB1 as described above.

Particularly in the third-described strain, ac
To explain that the high expression of yB1 is not due to the action of transcriptional activation sequences such as promoters and enhancers in the acyB2 coding region, but because the protein encoded by acyB2 functions by being translated. It is suitable. In addition, observing the recovery of high expression of acyB1 when the normal acyB2 function is given again to the strain that lost the acyB2 function is also one means for understanding the function of acyB2. For example, the first described Streptomyces thermotolerer (Streptomyces thermotoler)
ans) acyB2 mutant strain by introducing the acyB2 gene linked to the plasmid into acyB2 mutant strain.
It is possible to see if a recovery of 1 is seen (Example 7).

In the loss-of-function strain of acyB2, acy
To evaluate the expression of B1, for example, after culturing in an appropriate medium as described in detail in Example 6, directly in a culture solution, or after collecting the cells and suspending them in an appropriate buffer, 4 " A 4 "-isovaleryl derivative (leucomycin A43) obtained by adding a macrolide antibiotic whose position is OH group type, such as leucomycin U, and carrying out a conversion reaction for a certain period of time
Can be carried out by separating and quantifying TLC or HPLC. Furthermore, the expression level of acyB1 is
It can also be determined by Northern hybridization using a probe that specifically pairs with acyB1 mRNA. This method for measuring acyB1 mRNA differs from the method for assaying the conversion activity of macrolide antibiotics described above in that it measures the enzymatic activity of acyB1, whereas this method for measuring acyB1 mRNA differs in that it measures the transcriptional activity of acyB1. It is also convenient to know whether the expression control of is established at the transcription level or in the process after transcription.

The present invention will be described in more detail below with reference to examples. These examples are merely illustrative of the invention and are not intended to limit the scope of the invention.

【Example】

Example 1 Construction of shuttle vector pSAN-lac HincI of plasmid pUC18 (Nippon Gene)
0.5 μg of I degradation product and 0.2 μg of BglII linker (Pharmacia) were added to TE buffer (10 mM Tris / HC
1, 1 mM EDTA, pH 8.0, dissolved in 45 μl and then 10 × ligation buffer (500 mM
Tris-HCl (pH 7.9), 100 mM MgC
l ₂ , 200 mM DTT, 10 mM ATP) 5 μl and T ₄ DNA ligase (Nippon Gene) 3 μl (90
0 unit) was added and incubated at 16 ° C. for 15 hours. After the reaction, the DNA was purified by phenol / chloroform extraction and ethanol precipitation, and then digested again with HincII to linearize the plasmid in which the BglII linker was not inserted. After the reaction, E. coli JM103 competent cells were transformed with half the reaction solution (10 μl). For the preparation and transformation of competent cells, refer to the manual of Maniatis et al. (Molecular Cloning, A Labo) throughout the following examples.
rally Manual, Cold Spring
Harbor Laboratory, New Yo
rk, USA).

The selection medium is 0.5 mM IPTG, 0.0
LB medium (1% bactotryptone, 0.5% yeast extract, 1% NaCl, 1.5% bacto agar) supplemented with 1% Xgal, 50 μg / ml ampicillin was used. Among the large number of colonies that appeared, those showing blue color, that is, β-galactosidase activated strain, were selected,
Moreover, HincII (SalI) site is BglII
The plasmid pUC-Bg having the structure substituted in the above was prepared in large scale according to the method described in the aforementioned manual.

Next, 0.5 μg of the AatII degradation product of this plasmid and 1.0 μg of the PstI degradation product of pIJ350 (described in Japanese Patent Application Laid-Open No. 2-72880) were dissolved in 23 μl of TE, and 10 × polymerase buffer (70 mM Tris-HCl (pH 7 .4), 500 mM NaCl, 70 m
MMgCl ₂ ) 3 μl, dNTP mixed solution (dATP,
dTTP, dGTP, dCTP 0.5 mM mixture)
3 μl, T ₄ DNA polymerase (1 μm from Toyobo Co., Ltd.)
1 (2.4 units) was sequentially added, and the mixture was incubated at 37 ° C for 30 minutes, and then DNA was purified by phenol / chloroform extraction and ethanol precipitation.
It was dissolved in 1. Further, to this solution, 5 μl of 10 × ligation buffer and 2 μl of T ₄ DNA ligase (60
0 unit) was added and incubated at 16 ° C. for 15 hours. After the reaction, E. coli JM103 competent cells were transformed with 10 μl and ampicillin was added (50 μl).
(g / ml) As a result of selection in LB medium, a large number of colonies were obtained.

A large amount of the extracted plasmid was prepared from a strain having the structure shown in FIG. This plasmid was named pSAN-lac. pSAN
-Lac is an E. coli vector pUC18 and Streptomyces lividans (Streptomyces).
lividans) and other actinomycetes vector pIJ35
It is a shuttle vector with both replicon 0. Furthermore, it is possible to use the E. coli multi-cloning site,
In addition, it is a plasmid with extremely high utility value because it can utilize a marker for inactivating insertion of β-galactosidase when Escherichia coli is used as a host.

Example 2 Method for Preparing acyB2 Fragment Streptomyces lividans containing the known plasmid pA0207
vidans) # 207 (Mikori Kenjoyori No. 1880) by the method of Hopwood and others [Genetic Manipulation of Streptomyces of Genetic Manipulation of of
Streptomyces): Laboratory Manual (A Laboratory Manual),
John Innes Foundation (John In
Plasmid Foundation pAO207 was extracted and purified in a large amount according to the "Nes Foundation"].

20 μg of this plasmid was added to SacI and Sp
It was completely digested with hI and then electrophoresed on a 0.8% agarose gel to give acyB1 and acyB2 shown in FIG. 3A.
A fragment of about 3.4 kb containing DNA was isolated. Next, the agarose gel containing this fragment was cut out, and GENE CLEANI was cut.
I (BIO101, La Jolla, Califor
a, USA) to give 1.
5 μg of DNA fragment was recovered and purified. On the other hand, the shuttle vector pSAN-lac2 described in Example 1 above.
After completely decomposing μg with SacI and SphI, the product was purified by phenol / chloroform extraction and ethanol precipitation, and then TE (10 mM Tris · HCl, 1 mMED
It was dissolved in 20 μl of TA, pH 8.0). 2μ of this solution
1 and the TE of SacI-SphI approximately 3.4 kb fragment described above.
20 μl (1 μg) of the solution was mixed, 2.5 μl of 10 × ligation buffer and 0.5 μl (150 units) of T ₄ DNA ligase (Nippon Gene) were further added, and the mixture was incubated at 16 ° C. for 15 hours. After the reaction, 1
E. coli JM103 competent cells were transformed with 5 μl.

The selection medium is 0.5 mM IPTG, 0.0
LB medium (1% bactotryptone, 0.5% yeast extract, 1% NaCl, 1.5% bacto agar) supplemented with 1% Xgal, 50 μg / ml ampicillin was used. Among the many colonies that appeared, one that did not show blue color, that is, a β-galactosidase-inserted inactive strain was selected, and the plasmid pMAB8 having the structure shown in FIG. 5 was cloned into the manual of Maniatis et al.
Ning, A Laboratory Manual,
Cold Spring Harbor Labora
Mass preparation according to the method described in Tory, New York, USA). Next, this plasmid 50
A fragment obtained by completely digesting μg with PstI and SphI and having a size of about 2.1 kb and containing acyB2 was separated by 0.8% agarose gel electrophoresis, and then GENE CLE.
About 5 after recovery and purification by ANII (BIO101)
μg of the acyB2 fragment shown in FIG. 3B was obtained.

Example 3 Construction of Deletion Plasmid (1) Construction of pMAB9 0.5 μg of a degradation product of MluI and SphI of pMAB8 was dissolved in 23 μl of TE, 10 μl polymerase buffer 3 μl, dNTP mixture 3 μl T ₄ DNA polymerase (Toyobo ( Strain) 1 μl (2.4 units) was added sequentially and incubated at 37 ° C. for 30 minutes to blunt the DNA ends, followed by phenol / chloroform extraction,
DNA was purified by ethanol precipitation and TE43 μl
Dissolved in. To this solution, 5 μl of 10 × ligation buffer, T ₄ DNA ligase (Nippon Gene) 2
μl (600 units) was added, and self-cyclization was performed at 16 ° C. for 15 hours.

After the reaction, 25 μl was used to prepare Streptomyces lividans TK24 (Streptomyces).
lividans TK24) protoplasts (1
X10 ⁹ cells) were subjected to transformation treatment. For details of transformation, the method described in the manual of Hopwood et al. Was followed. From the Streptomyces lividans transformants obtained as a result, the strain having pMAB9 shown in FIG. 6 was isolated. pMAB9 is an M containing pMAB8 to acyB2
This is a plasmid in which the approximately 1.3 kb fragment of luI-SphI was deleted.

(2) Construction of pMAB10 It was constructed by the method described in Example 1. pSAN
-Lac 2 μg was completely digested with PstI and SphI. After the DNA was purified by phenol / chloroform extraction and ethanol precipitation, 20 μl of T
Dissolved in E. 15 μ in 2 μl (0.2 μg) of this solution
1 (1 μg) of acyB2 fragment in TE solution (see Example 2) is mixed, 2 μl of 10 × ligation buffer and 1 μl (300 units) of T ₄ DNA ligase (Nippon Gene) are added, and the mixture is incubated at 16 ° C. for 15 hours. did. After the reaction, 10 μl was used to streptomyces lividans TK24 (Streptom).
yces lividans TK24) was carried out in the same manner as in the above-mentioned pMAB9, and among the resulting Streptomyces lividans transformants, FIG.
A strain having the plasmid pMAB10 shown in 1 was obtained. pMAB10 is a from the insert fragment of pMAB8.
SacI-PstI1.1k containing most of cyB1
It has the same function as the plasmid in which the b fragment was deleted.

Example 4 Induction of frameshift mutation in pMAB8 PstI degradation product, MluI degradation product, and Afl of pMAB8
According to the reaction conditions described in the pMAB9 construction method of Example 3, 0.5 μg of each of the II degradation products was blunted with T ₄ DNA polymerase and self-circularized with T ₄ DNA ligase, and finally Streptomyces.・ Libido TK24 (Streptomyce)
S. lividans TK24) was transformed. Among the transformants obtained as a result, Ps
Plasmid pMAB8 starting from the one using tI degradation product
A strain having the plasmid pMAB8ΔAfl was isolated from those obtained by using the degradation products of ΔPst and MluI as the materials and those obtained by using the degradation products of the plasmids pMAB8ΔMlu and AflII. pMAB8ΔPst is a plasmid in which a frameshift mutation was induced as a result of the PstI site originally present on the acyB1 translation region being destroyed by blunting, and similarly pMAB8ΔMlu and pMAB8Δ.
Afl is also Mlu originally present in the acyB1 translation region.
This is a plasmid having a frameshift mutation due to the disruption of I and AflII (FIG. 8).

Example 5 Construction of pMAB-B2 pIJ702 (Streptomyces lividans ATC)
C35287 (Streptomyces lived
ans ATCC35287)) PstI and SphI degradation product 0.2 μg and acyB2 fragment 1 μm
g was dissolved in 17 μl of TE, 10 × ligation buffer-2 μl and 1 μl (300 units) of T ₄ DNA ligase (Nippon Gene) were added, and the mixture was incubated at 16 ° C. for 15 hours, and then strept was performed according to the method described in Example 3. Myces Rebidance TK24 (St
reptomyces lividans TK24)
Was transformed. From the obtained transformants, a strain that does not produce black pigment (melanin), that is, a strain in which the tyrosinase gene has been inactivated by insertion was selected. As a result, a strain having the plasmid pMAB-B2 shown in FIG. 9 was isolated. .
A large amount of this plasmid was prepared by treating this strain by the method described in Example 1.

Example 6 Measurement of acyB1 expression level (1) Leucomycin U conversion assay Streptomyces lividans recombinant and streptosome used for measurement of acyB1 expression level by macrolide-4 "-O-acyltransferase activity Myces Thermo Tolerance TSB (triptycase
Soy broth) 50 ml (containing 5 μg / ml of thiopeptin) was shake-cultured at 28 ° C. for 3 days, and then the cells were collected to give 1 ml of assay buffer (1 ml per 0.2 g of wet cell weight).
00 μg / ml leucomycin U, 0.5 MTES (p
H7.0), 200 μg / ml CoCl ₂ ) was added and well suspended. Inject 1 ml of this suspension into a test tube,
Incubated for 1 hour at 0 ° C. under shaking conditions. Then, the mixture was extracted with 1 ml of ethyl acetate, the ethyl acetate was distilled off, and the residue was dissolved in 50 ml of 2% acetonitrile. Using this solution as a sample, HPLC was performed under the following conditions to analyze the converted product.

Model: Hitachi L-6000 Column: Shimadzu shim pack CLC-ODS0.
15 m × 6.0 mmφ Solvent: 0.85 M NaClO ₄ (pH 2.5): Acetonitrile = 18: 15 Column temperature: 40 ° C. Detection: Absorption at wavelength 230 nm Injection amount: 10 μl As a result, leucomycin U used as a conversion substrate is The retention time was 3.50 minutes. In addition, leucomycin U-
4 "-O- isovaleryl embodying (leucomycin _A 3)
Had a retention time of 11.35 minutes. After sample analysis,
The conversion rate was calculated from these two peak area values.

FIG. 8 shows plasmids in which deletion mutations and frame shift mutations have been introduced, and pMAB8-transformed Streptomyces lividans TK24 as a control.
(Streptomyces lividans TK
The conversion rate of 24) is shown. According to this, it was shown that the expression of acyB1 was remarkably decreased in the recombinants having acyB2 mutation, regardless of the deletion mutation and the frameshift mutation.

(2) Northern hybridization Streptomyces thermotolerance ATCC114
16 (Streptomyces thermomotor
erans ATCC11416) and pMAB8,
Streptomyces lividans TK24 (St transformed with pMAB9, pMAB10, pAO207, etc.
reptomyces lividans TK24)
25 ml of TSB was inoculated and precultured at 28 ° C. for 3 days. 2 ml of this culture broth was added to 40 ml of YEME medium (described in the manual of Hopwood et al.), And the culture was continued again for 3 days. When culturing the recombinant, 5 μg /
ml of thiopeptin was added. After culturing, collect the cells,
Total RNA was extracted by treating the cells according to the method described in Hopwood et al. Then, 10 μg of each sample was subjected to ethanol precipitation of total RNA, followed by the method described by Davis et al. (Basic Methods in Molecule).
ar Biology, Elevievier Scie
nce Publishing Co. , Inc. Ne
w. York, USA).

The probe was nick-translated with α- [ ³² P] dCTP (Amersham Japan). A 0.5 kb acyB1 fragment of PstI-PvuII was used. For autoradiography, X
Using wire film (Kodak XAR-5), -70 ° C
Exposed for 2.5 hours. As a result, mRN of acyB1
The amount of A depends on the parent strain Streptomyces thermotoler.
erans) revealed that there was a marked increase in recombinants containing both acyB1 and acyB2.
However, among the recombinants, mRNA was not detected at all in the acyB1 deletion mutation, and significantly decreased in those having the acyB2 deletion mutation (FIG. 10). From this, the high expression of acyB1 by acyB2 is m
It can be seen that it occurs at the transcription level of RNA.

Example 7 Acquisition of Streptomyces thermotolerance acyB2 mutant and transformation with pMAB-B2 Streptomyces thermotolerance ATCC114
16 (Streptomyces thermoto
spell formation by culturing 7 days at 28 ° C. on SSYm medium (soluble starch 0.4%, yeast extract 0.4%, malt extract 1.0%, bacto agar 1.5%, pH 7.2). I went. Spores were suspended in a sterilized 20% glycerol aqueous solution to give a spore suspension (10 ⁶ cells / μl). 10 μl of this suspension is 1 m
l of _{0.1M KH 2 PO 4 -NaOH (pH8} .
0), and then 200 μg of NTG (N-methyl N′-nitro-N-nitrosoguanidine) was added, and 3
It was left at 0 ° C. for 1 hour. 5 ml of this spore 0.1M
After washing 3 times with KH ₂ PO ₄ -NaOH (pH 8.0), it was suspended again in 1 ml. 10-10 this suspension
^It was diluted ^3- fold, spread on ISP-2 medium, and cultured at 28 ° C. for 5 days. The emerging colonies were separated and the conversion activity of leucomycin U was measured (see Example 6). As a result, acyB2 mutant strains Streptomyces thermotolerance 2257 and Streptomyces thermotolerance 2398 were obtained. In these strains,
The function of cyB2 was lost, and the acylation activity of macrolide was remarkably reduced. Protoplasts of these two mutant strains were prepared according to the method described by Hopwood et al., And transformed with the plasmid pMAB-B2 constructed by the method described in Example 5. The results of investigating the conversion activity of leucomycin U on the obtained transformant are shown in Table 1.

[Table 1] As is clear from Table 1, the transformant activity of leucomycin U in the transformant was 1/3 and 2.5 of that of Streptomyces thermotolerance ATCC11416, respectively.
It was about twice as recovered.

[Brief description of drawings]

FIG. 1 is a nucleotide sequence of DNA containing the gene acyB2.

FIG. 2 shows the amino acid sequence of the protein encoded by the gene acyB2.

FIG. 3 is a restriction map of a DNA fragment containing acyB1 and acyB2, and FIG. B is acyB2.
3 is a restriction map of a DNA fragment containing

FIG. 4 is a restriction enzyme breakpoint and function map of plasmid pSAN-lac.

FIG. 5 is a restriction map and functional map of plasmid pMAB8.

FIG. 6 is a restriction map and functional map of plasmid pMAB9.

FIG. 7 is a restriction enzyme breakpoint and function map of plasmid pMAB10.

FIG. 8 is a diagram illustrating functions of acyB1 and acyB2.

FIG. 9 is a restriction map and functional map of plasmid pMAB-B2.

FIG. 10 shows the results of Northern hybridization using a 0.5 kb DNA fragment of acyB1 as a probe.

[Sequence list]

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[Procedure amendment]

[Submission date] October 1, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Correction content]

[Document name] Statement

Title: Method for high expression of acylating enzyme gene of macrolide antibiotic

[Claims]

[Chemical 1] A method for highly expressing the 4'-position acylating enzyme activity of a macrolide antibiotic, which comprises introducing the DNA fragment represented by:

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a DNA fragment encoding a protein having an action of enhancing the expression efficiency when an enzyme gene for acylating the 4 "-position of a macrolide antibiotic is introduced into a host and expressed. Regarding usage.

Macrolide antibiotics (14-membered ring and 1
The 6-membered ring is a useful antibiotic that is widely used as a medicine and a veterinary medicine (for domestic animals and fisheries). Acylation of the 4 "hydroxyl group of 16-membered macrolide antibiotics increases antibacterial activity by S. Omura et al. [Journal of Antibiotics].
Antibiotics) 28, 401-433 (1980)] and R.A. Okamoto et al. [Journal of Antibiotics
f Antibiotics) Volume 27, 542-544
Page (1979)]. One of the 16-membered macrolide antibiotics, tylosin-producing bacterium Streptomyces fladiae (Streptomyc)
es fradiae) produces only unacylated at the 3 and 4 "positions, i.e. the -OH form. To acylate this, once tylosin has been separated, a chemical method or biosynthesis is performed. Recently, a method for directly producing a 4 "-acylated tylosin by a genetic engineering method has been disclosed (JP-A-2-728).
No. 80).

The present invention enhances the expression of the 4 "-position acylation product in the case of producing a 4" -position acylation product by using the macrolide antibiotic 4 "-position acylating enzyme gene, and highly efficiently Which encodes a protein capable of producing
A method of using the A fragment is provided.

[0004]

2. Description of the Related Art The following macrolide antibiotics have been known, for example. Tylosin,

[Chemical 2] Angoramycin,

[Chemical 3] Spiramycin,

[Chemical 4] Leucomycin,

[Chemical 5] Deltamycin,

[Chemical 6] As a genetically engineered method for acylating the 4 "-position of these macrolide antibiotics, Janet Kay Ep et al. (JP-A-2-13380) encodes 4" -O-isovaleryl acylase. The use of DNA fragments containing In this method, a DNA fragment encoding a 4 "-position acylating enzyme is inserted into a vector plasmid for the purpose of genetically acylating a macrolide antibiotic having an 4" -OH position, and the host is transformed with the plasmid. Then, the obtained transformant is cultured. However, according to this method, the expression of the gene was weak, and 4 "-O-
It was not sufficient for the industrial production of isovalerylated macrolides.

[0005]

DISCLOSURE OF THE INVENTION The present invention aims at enhancing the expression of a 4 "-position acylating enzyme gene in a microorganism having a macrolide antibiotic 4" -position acylating enzyme gene. It is to provide a method for efficiently producing a ride antibiotic.

[0006]

[Means for Solving the Problems] The inventors of the present invention have earnestly studied to industrially and economically produce a 4 "-position acylated macrolide by expressing a 4" -acylation enzyme gene of a macrolide antibiotic. As a result of repeated studies on the expression mechanism of the 4 "-position acylase gene, the inventors independently isolated acyB2 DNA fragment (Japanese Patent Laid-Open No. 7288/1990).
No. 0) is deeply involved in the expression of the 4 "-position acylase gene, and it was found that the acyB2 DNA fragment significantly promotes the expression, and the present invention has been completed.

Thus, according to the present invention, Streptomyces thermotolerance
A size of about 2.1 kilobases (kb) derived from the thermotolerans ATCC11416 strain and a DNA fragment specified by the nucleotide sequence shown in FIG. 1 is provided. DNA fragment acyB2 of the present invention or its D
The NA restriction fragment is useful for industrially producing a 4 "-acylated tylosin using a 4" -acylase gene, and is a recombinant DNA for a microorganism producing a macrolide antibiotic of Streptomyces. It is particularly useful in that it allows industrial use of the technology. Hereinafter, the production of an acylated macrolide antibiotic by a transformant using a DNA fragment containing the gene acyB2 of the present invention will be described in more detail.

Any strain of the genus Streptomyces, which is the source of the DNA fragment containing the gene acyB2 of the present invention, may be any strain as long as it is capable of producing a macrolide antibiotic 4′-position acylating enzyme. Can be used, for example, Streptomyces kitasatoens
is), Streptomyces narvonensis baru
Josamy Thetics (Streptomyces n)
arbonensis var josamyceti
cus), Streptomyces hygroscopic
us), Streptomyces platensis (Stre
ptomyces platensis, Streptomyces
s albireticuli), Streptomyces c.
inerochromogenes), Streptomyces jakartensis (Streptomyces)
djakartensis), Streptomyces fur
didicicus), Streptomyces macrosporus
oreus), Streptomyces tendae (Str)
eptomyces tendae, Streptomyces thermotolerance
thermotolerans, Streptomyces delta
e), etc., among which Streptomyces
Thermotolerance is preferred.

<Preparation of acyB2 fragment> ac of the present invention
The yB2-containing fragment can be obtained from the known plasmid pAO207. This plasmid was obtained from the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, Microtechnology Research Institute No. 1880 (FE
It can be extracted from the strain deposited as RM BP-1880). The above strain was aerobically cultured at 28 ° C. to obtain the strain, and the method of Hopwood et al. [Genetic Manipulation of Streptomyces (Genetic Manipulation) was used.
of Streptomyces: A Laboratory Manual (A Laboratory Manual)
l), John Innes Foundation (John
The plasmid pAO207 can be prepared by treatment according to the Inns Foundation, Norwich, UK, 1985]. Furthermore, of the fragments inserted into pAO207, the approximately 3.4 kb SphI-SacI fragment containing acyB1 and acyB2 shown in FIG. 3A can be subcloned by an appropriate method well known in genetic engineering.

Examples of the vector used at this time include E. coli vector pUC18 and actinomycete vector pI.
Various materials such as J702 can be used, but especially Escherichia coli-actinomycete shuttle vector pUJ350 (JP-A-2
-72880) and pSAN-lac (Example 1) are convenient. The fragment subcloned in this way can be prepared in large quantities as a plasmid. Next, in order to obtain a fragment containing these plasmids acyB2, an appropriate restriction enzyme capable of cutting out the fragment may be selected. For example, from the restriction enzyme map of the inserted fragment as shown in FIG. As shown in PstI-Sp
By digesting with hI, an acyB2 fragment with a size of about 2.1 kb (kilobase) can be obtained as shown in FIG. 3B.

In this acyB2 fragment, the DNA sequence up to the 1630th position with the first base (cytosine) at the PstI site as 1 is shown in FIG. Furthermore, the amino acid sequence of the protein encoded from the 450th position to the 1610th position of this fragment is shown in FIG.

<Functional analysis of acyB2> In general, in order to investigate whether the expression of a gene in the same organism is regulated by other factors such as suppression or promotion, the factor having the regulatory function is functioned by some method. It seems that the most appropriate method is to investigate whether or not the expression is deregulated in the case where it is abolished. From this point of view, to explain that high expression of the macrolide-4 ″ -acylating enzyme gene acyB1 is achieved by the protein encoded by acyB2, the expression of acyB1 in the case where the protein is made non-functional can be explained. This can be proved by looking at the decrease, and there are several possible means for disabling the protein encoded by acyB2, but there are three main ones.

First, Streptomyces thermotolerance (St) which is the origin of acyB1 and acyB2
reptomyces thermotoleran
acy by chemically modifying s) with NTG or the like
This is the acquisition of a mutant strain in which the B2 function is lost. Second, acy
A recombinant plasmid containing B1, for example, a plasmid obtained by deleting most of the acyB2 region from a plasmid such as pMAB8 described in Example 2 was transformed with Streptomyces lividans (Streptt.
omyces lividans) TK24. Third, in the pMAB8 recombinant plasmid described above, ac
Streptomyces lividans transformed with a frameshift mutation in the protein coding region of yB2
It is TK24. Therefore, these strains are suitable for studying the action of acyB2 on acyB1 as described above.

Particularly in the third-described strain, ac
To explain that the high expression of yB1 is not due to the action of transcriptional activation sequences such as promoters and enhancers in the acyB2 coding region, but due to the fact that the protein encoded by acyB2 is translated and functions. It is suitable. In addition, when a strain that has lost acyB2 function is given normal acyB2 function again, ac
Looking at the recovery of high expression of yB1 is also one means for understanding the function of acyB2. For example, the first explained Streptomyces thermotolerance
It is possible to examine whether or not recovery of acyB1 is observed by introducing the acyB2 gene linked to the plasmid into the acyB2 mutant strain of Ptomyces thermotolerans (Example 7).

In the loss-of-function strain of acyB2, acy
To evaluate the expression of B1, for example, after culturing in an appropriate medium as described in detail in Example 6, directly in a culture solution, or after collecting the cells and suspending them in an appropriate buffer, 4 " A 4 "-isovaleryl derivative (leucomycin A3) obtained by adding a macrolide antibiotic such as leucomycin U whose position is an OH group type and performing a conversion reaction for a certain period of time is separated and quantified by TLC or HPLC. Can be done by Furthermore, the expression level of acyB1 is a
It can also be determined by Northern hybridization using a probe that specifically pairs with cyB1 mRNA. This method for measuring acyB1 mRNA differs from the method for assaying the conversion activity of macrolide antibiotics described above in that it measures the enzymatic activity of acyB1, whereas this method for measuring acyB1 mRNA differs in that it measures the transcriptional activity of acyB1. It is also convenient to know whether the expression control of is established at the transcription level or in the process after transcription.

The present invention will be described in more detail below with reference to examples. These examples are merely illustrative of the invention and are not intended to limit the scope of the invention.

Example 1 Construction of shuttle vector pSAN-lac HincI of plasmid pUC18 (Nippon Gene)
0.5 μg of I degradation product and 0.2 μg of BglII linker (Pharmacia) were added to TE buffer (10 mM Tris / HC
1, 1 mM EDTA, pH 8.0, dissolved in 45 μl and then 10 × ligation buffer (500 mM
Tris-HCl (pH 7.9), 100 mM MgC
l ₂ , 200 mM DTT, 10 mM ATP) 5 μl and T ₄ DNA ligase (Nippon Gene) 3 μl (90
0 unit) was added and incubated at 16 ° C. for 15 hours. After the reaction, the DNA was purified by phenol / chloroform extraction and ethanol precipitation, and then digested again with HincII to linearize the plasmid in which the BglII linker was not inserted. After the reaction, E. coli JM103 competent cells were transformed with half the reaction solution (10 μl). For the preparation and transformation of competent cells, the manuals of Maniatis et al. (Molecular Cloning, A Labo) are used throughout the following examples.
rally Manal, Cold Spring
Harbor Laboratory, NewYor
k, USA).

The selection medium is 0.5 mM IPTG, 0.0
LB medium (1% bactotryptone, 0.5% yeast extract, 1% NaCl, 1.5% bacto agar) supplemented with 1% Xgal, 50 μg / ml ampicillin was used. Among the large number of colonies that appeared, those showing blue color, that is, β-galactosidase activated strain, were selected,
Moreover, HincII (SalI) site is BglII
The plasmid pUc-Bg having the structure substituted in was prepared in large scale according to the method described in the above-mentioned manual.

Next, 0.5 μg of the AatII degradation product of this plasmid and 1.0 μg of the PstI degradation product of pIJ350 (described in Japanese Patent Application Laid-Open No. 2-72880) were dissolved in 23 μl of TE, and 10 × polymerase buffer (70 mM Tris HCl (pH 7. 4), 500 mM NaCl, 70 mM
MgCl ₂ ) 3 μl, dNTP mixed solution (dATP, d
0.5 mM each of TTP, dGTP, and dCTP) 3
μl, T ₄ DNA polymerase (Toyobo Co., Ltd. 1 μl)
(2.4 units) were sequentially added, and the mixture was incubated at 37 ° C for 30 minutes, and then the DNA was purified by phenol / chloroform extraction and ethanol precipitation, and then TE43 µl
Dissolved in. Furthermore, 5 μl of 10 × ligation buffer and 2 μl (600 units) of T ₄ DNA ligase were added to this solution, and the mixture was incubated at 16 ° C. for 15 hours. After the reaction, E. coli JM103 competent cells were transformed with 10 μl and ampicillin was added (50 μg).
/ Ml) As a result of selection in LB medium, a large number of colonies were obtained.

A large amount of the extracted plasmid was prepared from a strain having the structure shown in FIG. This plasmid was named pSAN-lac. pSAN
-1ac is an E. coli vector pUCl8 and Streptomyces lividans (Streptomyces).
lividans) and other actinomycetes vector pIJ350
It is a shuttle vector having both replicon. Further, it is a plasmid having a very high utility value since it can use the multi-cloning site of Escherichia coli and can utilize the insertion inactivation marker of β-galactosidase when E. coli is used as a host.

Example 2 Method for Preparing acyB2 Fragment Streptomyces lividans strain carrying the known plasmid pAO207
vidans) # 207 (Mikori Kenjoyori No. 1880) by the method of Hopwood and others [Genetic Manipulation of Streptomyces of Genetic Manipulation of of
Streptomyces: A Laboratory Manual, John Innes Foundation (John Inn)
es Foundation)] and a large amount of the plasmid pAO207 was extracted and purified.

20 μg of this plasmid was added to SacI and Sp
It was completely digested with hI and then electrophoresed on a 0.8% agarose gel to give acyB1 and acyB2 shown in FIG. 3A.
A fragment of about 3.4 kb containing DNA was isolated. Next, the agarose gel containing this fragment was cut out, and GENE CLEANI was cut.
I (BIO101, La Jolla, Califor
nia, USA) to recover and purify 1.5 μg of DNA fragments from the gel pieces. On the other hand, the shuttle vector pSAN-la described in Example 1 above.
After c2 μg was completely decomposed with SacI and SphI, and purified by phenol / chloroform extraction and ethanol precipitation, TE (10 mM Tris · HCl, 1 mM
It was dissolved in 20 μl of DTA, pH 8.0. This solution 2
μl and the T of the above SacI-SphI approximately 3.4 kb fragment
20 μl (1 μg) of E solution was mixed, 2.5 μl of 10 × ligation buffer and 0.5 μl (150 units) of T ₄ DNA ligase (Nippon Gene) were further added, and the mixture was incubated at 16 ° C. for 15 hours. After the reaction
E. coli JM103 competent cells were transformed with 15 μl.

The selection medium is 0.5 mM IPTG, 0.0
LB medium (1% bactotryptone, 0.5% yeast extract, 1% Nacl, 1.5% bacto agar) supplemented with 1% Xgal, 50 μg / ml ampicillin was used. Among the many colonies that appeared, one that did not show blue color, that is, a β-galactosidase-inserted inactive strain was selected, and the plasmid pMAB8 having the structure shown in FIG. 5 was cloned into the manual of Maniatis et al.
Ning, A Laboratory Manual,
Cold Sprig Harbor Laborat
mass preparation according to the method described in (Ory, New York, USA). Next, this plasmid 50μ
A fragment obtained by completely decomposing g with PstI and sphl was about 2.1 kb and containing acyB2 was separated by 0.8% agarose gel electrophoresis, followed by GENE CLEA.
About 5μ after recovery and purification with NII (BIO101)
g of acyB2 fragment shown in Figure 3B was obtained.

Example 3 Construction of Deletion Plasmid (1) Construction of pMAB9 0.5 μg of a degradation product of MluI and sphI of pMAB8 was dissolved in 23 μl of TE, 10 μl polymerase buffer 3 μl, dNTP mixture 3 μl T ₄ DNA polymerase (Toyobo ( Strain)) 1 μl (2.4 units) were sequentially added and incubated at 37 ° C. for 30 minutes to blunt the DNA ends. After that, phenol / chloroform extraction,
DNA was purified by ethanol precipitation and TE43 μl
Dissolved in. 5 μl of 10 × ligation buffer and 2 μ of T ₄ DNA ligase (Nippon Gene) were added to this solution.
1 (600 units) was added, and self-cyclization was performed at 16 ° C. for 15 hours.

After the reaction, 25 μl was used to prepare Streptomyces lividans TK24 (Streptomyces).
lividans TK24) protoplasts (1
X10 ⁹ cells) were subjected to transformation treatment. For details of transformation, the method described in the manual of Hopwood et al. Was followed. From the Streptomyces lividans transformants obtained as a result, the strain having pMAB9 shown in FIG. 6 was isolated. pMAB9 is an M containing pMAB8 to acyB2
This is a plasmid in which the approximately 1.3 kb fragment of luI-SphI was deleted.

(2) Construction of pMAB10 It was constructed by the method described in Example 1. pSAN
-Lac 2 μg was completely digested with PstI and SphI. After the DNA was purified by phenol / chloroform extraction and ethanol precipitation, 20 μl of T
Dissolved in E. 15 μ in 2 μl (0.2 μg) of this solution
1 (1 μg) of acyB2 fragment in TE solution (see Example 2) is mixed, 2 μl of 10 × ligation buffer and 1 μl (300 units) of T ₄ DNA ligase (Nippon Gene) are added, and the mixture is incubated at 16 ° C. for 15 hours. did. After the reaction, 10 μl of the Streptomyces lividans TK24 (Streptomy) was used.
ces lividans TK24) was transformed in the same manner as the above-mentioned pMAB9, and from the resulting Streptomyces lividans transformants, a strain having the plasmid pMAB10 shown in FIG. 7 was obtained. p
MAB10 is acyB derived from the insert of pMAB8.
It has the same function as the plasmid in which the SacI-PstI 1.1 kb fragment containing most of 1 was deleted.

Example 4 Induction of frameshift mutation in pMAB8 PstI degradation product, MluI degradation product, and Afl of pMAB8
According to the reaction conditions described in the pMAB9 construction method of Example 3, 0.5 μg of each of the II degradation products was blunted with T ₄ DNA polymerase and self-circularized with T ₄ DNA ligase, and finally Streptomyces.・ Libido TK24 (Streptomyces)
lividans TK24) was transformed. From the transformants obtained as a result, those obtained by using PstI degradation products as materials were selected from plasmid pMAB8ΔPs.
A strain having the plasmid pMAB8ΔAfl was isolated from those obtained by using the degradation products of t and MluI as the materials and those obtained by using the degradation products of the plasmids pMAB8ΔMlu and AflII. pMAB8ΔPst is a plasmid in which a frameshift mutation was induced as a result of the PstI site originally present on the acyB1 translation region being destroyed by blunting. Similarly, pMAB8ΔMlu and pMAB8ΔAf
l is also a plasmid in which a frameshift mutation occurred due to the disruption of MluI and AflII originally present on the acyB1 translation region (FIG. 8).

Example 5 Construction of pMAB-B2 pIJ702 (Streptomyces lividans ATC)
C35287 (Streptomyces lived
ans ATCC35287)) PstI and SphI degradation product 0.2 μg and acyB2 fragment 1 μm
g was dissolved in 17 μl of TE, 2 μl of 10 × ligation buffer and 1 μl (300 units) of T ₄ DNA ligase (Nippon Gene) were added, and the mixture was incubated at 16 ° C. for 15 hours. Seth Rebidance TK24 (st
reptomyces lividans TK24)
Was transformed. From the obtained transformants, a strain that does not produce black pigment (melanin), that is, a strain in which the tyrosinase gene has been inactivated by insertion was selected. As a result, a strain having the plasmid pMAB-B2 shown in FIG. 9 was isolated. .
A large amount of this plasmid was prepared by treating this strain by the method described in Example 1.

Example 6 Measurement of acyB1 expression level (1) Leucomycin U conversion assay Streptomyces lividans recombinant and streptosome used for measurement of acyB1 expression level by macrolide-4 "-O-acyltransferase activity Myces Thermo Tolerance TSB (triptycase
Soy pros) 50 ml (containing 5 μg / ml of thiopeptin) was shake-cultured at 28 ° C. for 3 days, and then the cells were collected and 1 ml of assay buffer (1 ml per 0.2 g of wet cell weight).
00 μg / ml leucomycin U, 0.5 MTES (p
H7.0), 200 μg / ml CoCl ₂ ) was added and well suspended. Inject 1 ml of this suspension into a test tube,
Incubated for 1 hour at 0 ° C. under shaking conditions. Then, the mixture was extracted with 1 ml of ethyl acetate, the ethyl acetate was distilled off, and the residue was dissolved in 50 ml of 2% acetonitrile. Using this solution as a sample, HPLC was performed under the following conditions to analyze the converted product.

Model: Hitachi L-6000 Column: Shimadzu shim pack CLC-ODS
0.15 m × 6.0 mmφ Solvent: 0.85 M NaClO ₄ (pH 2.5): Acetonitrile = 18: 15 Column temperature: 40 ° C. Detection: Absorption at wavelength 230 nm Injection amount: 10 μl As a result, leucomycin used as a conversion substrate U had a retention time of 3.50 minutes. In addition, leucomycin U-
4 "-O- isovaleryl embodying (leucomycin _A 3)
Had a retention time of 11.35 minutes. After sample analysis,
The conversion rate was calculated from these two peak area values.

FIG. 8 shows plasmids in which deletion mutations and frame shift mutations have been introduced, and pMAB8-transformed Streptomyces lividans TK24 as a control.
(Streptomyces lividans TK
The conversion rate of 24) is shown. According to this, it was shown that the expression of acyB1 was remarkably reduced in the recombinants having a mutation in acyB2 regardless of the deletion mutation and the frameshift mutation.

(2) Northern hybridization Streptomyces thermotolerance ATCC114
16 (Streptomyces thermomotor
erans ATCC11416) and pMAB8,
Streptomyces lividans TK24 (St transformed with pMAB9, pMAB10, pAO207, etc.
25 ml of TSB was inoculated with reptomyces lividans TK24) and precultured at 28 ° C. for 3 days. 2 ml of this culture broth was added to 40 ml of YEME medium (described in the manual of Hopwood et al.), And the culture was continued again for 3 days. When culturing the recombinant, 5 μg /
ml of thiopeptin was added. After culturing, collect the cells,
Total RNA was extracted by treating the cells according to the method described in Hopwood et al. Next, 10 μg of each sample was subjected to ethanol precipitation of total RNA, followed by the method described by Davis et al. (Basic Methods in Molecule).
ar Biology, Elevievier Scie
nce Publishing Co. , Inc ,. N
Northern hybridization was performed according to ew York, USA).

The probe was nick-translated with α- [ ³² P] dCTP (Amersham Japan). A 0.5 kb acyB1 fragment of PstI-PvuII was used. For autoradiography, X
Using wire film (Kodak XAR-5), -70 ° C
Exposed for 2.5 hours. As a result, mRN of acyB1
The amount of A depends on the parent strain, Streptomyces thermotolerance.
erans) revealed that there was a marked increase in recombinants containing both acyB1 and acyB2.
However, among the recombinants, mRNA was not detected at all in the acyB1 deletion mutation, and that in the acyB2 deletion mutation was significantly decreased (FIG. 10). From this, the high expression of acyB1 by acyB2 is m
It can be seen that it occurs at the transcription level of RNA.

Example 7 Acquisition of Streptomyces thermotolerance acyB2 mutant and transformation with pMAB-B2 Streptomyces thermotolerance ATCC114
16 (Streptomyces thermomotor
erans ATCC11416) on SSYM medium (soluble starch 0.4%, yeast extract 0.4%, malt extract 1.0%, bacto agar 1.5%, pH 7.2).
After culturing at 8 ° C. for 7 days, sporulation was performed. 20 sterilized
% Glycerol aqueous solution to suspend the spores (1
⁶ cells / μl). 10 μl of this suspension was added to 1 ml of 0.1 M KH ₂ PO ₄ —NaOH (pH 8.0), and then NTG (N-methyl-N′-nitro-N) was added.
-Nitrosoguanidine) (200 μg) was added, and the mixture was left at 30 ° C. for 1 hour. Add 5 ml of 0.1 M KH ₂ to the spores.
After washing 3 times with PO ₄ -NaOH (pH 8.0), the cells were suspended again in 1 ml. This suspension was diluted 10 to 10 ³ times, spread on an ISP-2 medium, and cultured at 28 ° C. for 5 days. The emerging colonies were isolated and leucomycin U
The conversion activity was measured (see Example 6). As a result, a
The cyB2 mutant strains Streptomyces thermotolerance 2257 and Streptomyces thermotolerance 2398 were obtained. In these strains, acyB
The function of 2 disappeared and the macrolide acylation activity was remarkably reduced. Protoplasts of these two mutant strains were prepared according to the method described in Hopwood et al., And the plasmid p constructed by the method described in Example 5 was prepared.
It was transformed with MAB-B2. The results of investigating the conversion activity of leucomycin U on the obtained transformant are shown in Table 1.

[Table 1] As is clear from Table 1, the transformant activity of leucomycin U in the transformed strain was 1/3 and 2.5 of that of Streptomyces thermotolerance ATCC11416, respectively.
It was about twice as recovered.

[0032]

[Sequence list]

[Brief description of drawings]

FIG. 1 is a nucleotide sequence of DNA containing the gene acyB2.

FIG. 2 shows the amino acid sequence of the protein encoded by the gene acyB2.

FIG. 3A is a D containing acyB1 and acyB2.
It is a restriction map of NA fragment, and FIG. B is a restriction map of a DNA fragment containing acyB2.

FIG. 4 is a restriction enzyme breakpoint and function map of plasmid pSAN-lac.

FIG. 5 is a restriction map and functional map of plasmid pMAB8.

FIG. 6 is a restriction map and functional map of plasmid pMAB9.

FIG. 7 is a restriction enzyme breakpoint and function map of plasmid pMAB10.

FIG. 8 is a diagram illustrating functions of acyB1 and acyB2.

FIG. 9 is a restriction map and functional map of plasmid pMAB-B2.

FIG. 10 is a diagram showing the results of Northern hybridization using a 0.5 kb DNA fragment of acyB1 as a probe.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display part (C12N 9/16 C12R 1: 465) (C12N 15/55 C12R 1: 465) (C12N 1/21 C12R 1: 465)

Claims (2)

[Claims] 1. The following nucleotide sequence is attached to a microorganism having a macrolide antibiotic 4′-acylase gene: A method for highly expressing the 4'-position acylating enzyme activity of a macrolide antibiotic, which comprises introducing the DNA fragment represented by: 2. The method for highly expressing the 4 "-position acylating enzyme activity of a macrolide antibiotic according to claim 1, wherein the macrolide antibiotic is selected from tylosin, spiramycin, angoramycin, leucomycin and deltamycin.