JPH07177892A - Gane coding 3-acylation enzyme of macrolide antibiotic substance - Google Patents

Abstract

PURPOSE:To obtain the subject gene capable of producing a 3-acylated macrolide antibiotic substance at a low cost. CONSTITUTION:This gene is a DNA fragment containing a gene acyA coding a 3-site acylating enzyme for a macrolide antibiotic substance, originated from a microbial strain belonging to the genus Streptomyces, having a size of about 1.8 kb or about 3.2 kb and characterized by a restriction enzyme map of the figure. The present invention also relates to a restriction DNA fragment obtained from the DNA fragment by a restriction enzyme treatment.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a DNA fragment containing a gene (referred to as "acyA") which imparts to a host microorganism an enzyme activity for acylating the 3-position of a macrolide antibiotic, which is D
The present invention relates to a recombinant DNA plasmid containing an NA fragment and a microorganism transformed with a plasmid incorporating a DNA fragment containing the acyA gene, and a method for producing a 3-position acylated macrolide antibiotic by the transformant.

Macrolide antibiotics (14-membered ring and 1
The 6-membered ring is a useful antibiotic that is widely used as a medicine and an animal drug (for domestic animals and fisheries). In recent years, various macrolide derivatives have been investigated due to the emergence of resistant bacteria. Positions 3 and 4 "of 16-membered macrolide antibiotics
The antibacterial activity is increased by acylation of the OH group at position R.
Okamoto et al. [Journal of Antibiotics] Vol. 27, 524-54
4 (1979)]. According to it, a derivative of tylosin acylated at the 3 and 4 "positions was prepared, and its drug efficacy was investigated against various bacteria resistant to macrolide antibiotics. Has been shown to have a higher pharmacological effect on these resistant bacteria than tylosin. However, the tylosin-producing strain produces only the -OH type at the 3rd and 4th positions, To acylate this, there was no suitable method other than the chemical method or biological method after once separating tylosin as described later.

The present invention provides a very industrially useful method for producing a strain which cannot originally directly produce such a 3-position acylation product of a macrolide antibiotic by using a genetic engineering method. It is a thing.

[0004]

2. Description of the Related Art Conventionally, the following macrolide antibiotics are known. Formula 1: Tylosin

[0005]

[Chemical 7]

Formula 2: Spiramycin I

[0007]

[Chemical 8]

Formula 3: Angoramycin

[0009]

[Chemical 9]

Formula 4: Leucomycin

[0011]

[Chemical 10]

Formula 5: Nidamycin

[0013]

[Chemical 11]

As a method for acylating these macrolide antibiotics, a chemical method is generally used [Omura et al., Pharmaceutical Journal, 106, 729-757 (198).
6)], it was difficult to efficiently acylate a specific position in the molecule. Therefore, a biological method was proposed [Japanese Patent Publication No. 53-15160]. The conventionally proposed biological method is a method of acylating tylosin using a microorganism having a acylation ability of tylosin, a culture solution or a treated product thereof, or an enzyme isolated from the microorganism or a content thereof. For example, Streptomyces
Streptomyces thermotolerans A
TCC 11416, Streptomyces hygroscopicus ATCC 2
1582, Streptomyces kitasatoensis (St
reptomyces kitasatoensis) IFO 13686 and the like, a culture medium or a cell extract is used to add and react with a macrolide antibiotic having OH type at the 3rd and / or 4 "positions as a substrate for conversion. However, according to this method, the macrolide antibiotic-producing bacterium and the microorganism having an acylating activity are separate, which is economically disadvantageous in industrial production.

[0015]

In order to biologically acylate a macrolide antibiotic of the 3 and / or 4 "OH type, as described above, first, the macrolide producing strain is cultured, After isolating the macrolide antibiotic,
It is converted by a microorganism having an acylating enzyme. 2
A step operation was necessary. Therefore, the present inventors have improved this, and for the purpose of producing an acylated macrolide by a macrolide-producing strain alone, isolate the enzyme gene from a microorganism having a macrolide acylating enzyme gene, It was considered that the gene could be introduced into a macrolide-producing strain by a genetic engineering method to directly produce an acylated macrolide. Among these enzyme genes, the 4 "-position acylating enzyme gene (acyB) has already been isolated and recently disclosed in, for example, JP-A-2-72880. However, on the other hand, 3
The position acylase gene has not been cloned so far. Therefore, in order to achieve the above-mentioned object, the present inventors have
An attempt was made to isolate the 3-position acylase gene (acyA).

[0016]

[Means for Solving the Problems] With the recent development of genetic engineering technology, a desired gene is taken out from a specific organism and ligated directly or into an appropriate vector,
It has become possible to introduce it into another desired microorganism [D.
A. Hopptudud et al .: Genetic manipulation of strepto
myces, The John Innes Foundation (1985)].

Therefore, the inventors of the present invention conducted diligent research to isolate the enzyme gene from the microorganisms having the acylating enzymes at the 3 and / or 4 "positions of the macrolide antibiotics as described above, and as a result, streptomycin was obtained. The present inventors have succeeded in isolating the 3-position acylase gene acyA from a certain strain belonging to the genus Cessella and completed the present invention.

Thus, according to the present invention, a restriction enzyme map derived from a strain belonging to the genus Streptomyces and having a size of about 1.8 kilobases (kb) and shown in FIG. 1 (A) is attached. Provided are a DNA fragment containing a gene acyA encoding a 3-position acylating enzyme of a macrolide antibiotic characterized by the DNA base sequence shown, and a DNA restriction fragment thereof.

According to the present invention, the size of the strain derived from the Streptomyces genus is about 3.2 kilobases (kb) and the restriction map shown in FIG. 1 (B) is attached. 3 of macrolide constituents characterized by
Containing the gene acyA encoding the acylating enzyme at position
An A fragment and a DNA restriction fragment by its restriction enzyme are provided.

The term "DNA restriction fragment by restriction enzyme" as used herein refers to DNA fragments of various sizes that have been decomposed by restriction enzymes into lengths necessary for expression of acylase activity. DNA fragment or D containing the gene acyA of the present invention
NA restriction fragments are useful in the commercial production of macrolide antibiotic derivatives of interest, such as 3-acylated tylosin, and commercial recombinant DNA technology against Streptomyces and other antibiotic-producing microorganisms. It is especially useful in that it can be used effectively.

Hereinafter, a DNA containing the gene acyA of the present invention
The production of the fragment, its properties, and the production of the acylated macrolide antibiotic by the transformant using the fragment will be described in more detail.

As a strain of the genus Streptomyces which is a source of the DNA fragment containing the gene acyA of the present invention, any strain can be used as long as it has the ability to produce the 3-position acylating enzyme of the macrolide antibiotic. For example, Streptomyces ambofaciens (Strept
omyces ambofaciens), Streptomyces kitasatoensis, Streptomyces narbonensyl var josamyceticus, S. hygroscopicu
s), Streptomyces platensis (S. platensi
s), S. albi
reticuli), S. cinerochromogenes, S. djakartensis, S. furdicidicus, S. furdicidicus, Streptomyces macrosporeus S. macrosporeu
s), Streptomyces tendae (S. tendae), Streptomyces thermotoler (S. thermotoler)
ans), Streptomyces deltae (S. deltae) and the like, among which Streptomyces thermotolerance is preferable.

The case where this preferred Streptomyces thermotolerance is used will be described below. Similarly, even when other strains are used, the gene encoding the 3-position acylating enzyme of the macrolide antibiotic ( acyA) can be isolated.

<Preparation of Genomic DNA> The donor of the DNA containing the acyA gene of the present invention, Streptomyces thermotolerans, is, for example, the American Type Culture Collection (The Amer).
ican Type Culture Collection) with deposit number ATCC1
It has been deposited under No. 1416 and is an easily available strain.

As described in detail in Example 1 below, for example, the above strain is aerobically cultivated at 28 ° C. to obtain the cells in the logarithmic growth phase, and the method of Hopwood et al. [Genetetic Manip. Genetic Manipulation of Streptomyces: a.
Laboratory Manual (A Laboratory Manual), John Innes Found
ation), Norwich, UK, 1985
Year], the genomic DNA containing the acyA gene can be prepared.

<Cloning of S. thermotolerans carbomycin biosynthesis related gene region> Generally, all or many genes related to the biosynthesis of antibiotics of the genus Streptomyces are collected in a specific region on genomic DNA or plasmid DNA ( The fact that they exist in the form of a "cluster" exists in the Proc. Natl. Acad. Sci. Proc. Of National Academy of Science by Fishman et al.
USA), 84, 8248 (1987), Binney (Bin
nie) 's Journal of Bacteriology (Journ
al of Bacteriology), 171, 887 (1989).
Year). Since the acyA gene of the present invention encodes an enzyme that acylates the hydroxyl group at the 3-position of the macrolide antibiotic carbomycin produced by S. thermotolerans, it can be regarded as belonging to one of biosynthesis-related genes, Therefore, it is considered to form a population with other biosynthesis related genes. So S. therm
In order to obtain the acyA gene of the present invention from otolerans genomic DNA, it is desirable to first clone the carbomycin biosynthesis gene region using a cosmid vector or the like. Because if the obtained clone is used as a sample for isolating acyA in a limited region, genome D
AcyA, which is much more efficient than using the whole NA as a sample
This is because it becomes a cloning means of. Specifically, for example, E. coli cosmid vector pHC79 with BamHI site and S.
The Sau 3AI site degradation product of thermotolerans genomic DNA is ligated and the in vitro packaging is performed on λ-farage to prepare a gene library of S. thermotolerans. Generation of a gene library using such a cosmid can be carried out by using a general genetic engineering technique which is frequently used in all organisms for the purpose of isolating a specific gene [for example, Maniati (Maniati
s) et al .: "Molecular cloning" 295-30
See page 7, Cold Spring Harbor Laboratory].

Further, a clone containing a carbomycin biosynthesis-related gene region is obtained from this gene library by preparing a probe from a gene belonging to a known carbomycin biosynthesis-related gene or a DNA fragment in the vicinity thereof. Colony hybridization is performed on a gene library using Escherichia coli HB101 as a host bacterium (many strains obtained by transducing Escherichia coli HB101 with cosmid DNA having a packaged S. thermotolerans genomic DNA fragment). Can be achieved by By this operation, a strain paired with the probe, that is, a strain having a cosmid in which a DNA fragment containing a carbomycin biosynthesis-related gene region is incorporated can be obtained. Cosmids derived from these strains have an insert fragment of up to about 45 kb, which contains a part or all of the DNA fragment as a probe and extends to the peripheral region. For example, one of these recombinant cosmids designated by the present inventors as pSE26 has an insertion fragment of about 45 kb containing an acyB fragment near one end of the insertion site (see Example 2 and C below). ). By the way, since there is an upper limit on the length of a DNA fragment that can be inserted into a cosmid, the entire region of the genes related to biosynthesis is not always incorporated into the inserted fragment in the screening using only one type of probe described above.
Therefore, as one method to solve this, for example, pSE2
6 A method of isolating a new DNA fragment of an appropriate size from a region as far as possible from the probe region in the insert and using this as a probe can be mentioned. Using this probe, colony hybridization can be carried out again from the S. thermotolerans library, and cosmid can be obtained from the paired strain. In this way, acyB-containing DNA used as the first probe, having a partial overlapping region with pSE26
Recombinant cosmids having a region far apart from the fragment in the insert fragment, such as pBM73 shown in item C of Example 2 below, can be isolated. In this way, it is possible in some cases to extend the cloned region by repeating this procedure, resulting in the cloning of carbomycin biosynthesis related genes from the S. thermomotolerans gene library. .

Thus, a new probe is prepared in a region distant from the original probe from the clone obtained by pairing with one probe, and a clone pairing with this probe is obtained again, or, depending on the case, By repeating this operation many times, the method of cloning the containing region of the gene of interest ranging from several tens of kilobases (kb) to over 100 kilobases is generally referred to as gene walking.
Can be carried out using a method widely known to those skilled in the art [eg, Tashiro et al.
Gene Research Method II, pp. 83-104 (1986), edited by the Japanese Biochemical Society]. <Cloning of acyA Gene with Vector Plasmid pIHY-17> The sheer cothmid pIHY-17 can be constructed as a vector for cloning and expressing acyA of the present invention (see FIG. 2). This vector is a vector having both functions of actinomycete vector pIJ922 and Escherichia coli cosmid pHC79, and can be specifically constructed by the method shown in Example 3 described later. Generally, by ligating an E. coli vector and an actinomycete vector with each other, a vector having an advantage of being introduced and replicable in both organisms,
That is, the shuttle vector can be easily constructed. In order to give the same advantage to this vector pIHY-17, the actinomycete vector pIJ922 and E. coli vector pH are used.
It incorporates each replicon of C79 and is given each selectable marker. In addition to this, pHC79
The packaging function derived from the cos site of is added.

By this function, even a large plasmid having a total vector length of up to about 50 kb can be easily introduced into host E. coli through in vitro packaging of λ phage. The above-mentioned function of pIHY-17 as a sheaturtimid is suitable for efficient cloning and expression of acyA. For example, ligating a cloned fragment derived from a gene region related to carbomycin biosynthesis to this vector,
The cos site can be used for in vitro packaging into λ phage. A large number of strains obtained by transducing this into Escherichia coli HB101 can be mixed to extract a plasmid. Using the function of pIJ922, this plasmid can be transformed into actinomycete S. lividans and the like. Among the obtained transformants, a strain to which acyA of the present invention is provided, that is, a strain having a macrolide 3-position acylating activity can be selected by a thin-layer chromatographic test, and as a result, acyA is cloned. Can be converted.

The above method is different from the case of using a vector dedicated to actinomycetes, and DN which has been reliably circularized and amplified by the operations of in vitro packaging and transduction.
It is excellent in that A can be provided for transformation of S. lividans.

<Subcloning of DNA Fragment Containing acyA Gene> By the method described above, the strain having the acyA activity of the present invention, for example, S. lividans 53A strain can be isolated. The plasmid, eg p53A, can also be isolated by treating this strain as described in Example 4. Whether the acyA of the present invention is present in the insert of the plasmid depends on whether the plasmid is S. livi.
This can be demonstrated by the macrolide 3-position acylation activity in the strain retransformed into dans. This insert fragment can be excised from the original plasmid using an appropriate restriction site and subcloned into E. coli vector pUC19 or the like. The thus obtained plasmids, such as pMAA2 and pMAA3 (see FIG. 4), can be prepared rapidly and easily in large quantities, and are used as suitable materials for analyzing the structure and expression function of the acyA gene of the present invention. be able to.

<Usefulness of DNA Restriction Fragment Containing acyA Gene> The 3-position acylating enzyme of the macrolide antibiotic expressed by using the acyA gene of the present invention is particularly useful for the acylation of 3-position of tylosin. However, it is not limited thereto, but it also has the 3-position acylating ability of other macrolide antibiotics such as spiramycin, angoramycin, leucomycin, and nidamycin.

Therefore, when the plasmid of the present invention in which the acyA gene is incorporated is introduced into these microorganisms capable of producing macrolide antibiotics, the macrolide antibiotics acylated at the 3-position are directly produced. It becomes possible.

It is also possible to introduce such a recombinant plasmid into a microorganism which does not produce macrolide antibiotics, and the transformed strain is cultivated in a medium containing macrolide antibiotics so that the 3-position is acylated. It is possible to produce macrolide antibiotics.

Furthermore, the 3-position acylating enzyme produced by the transformant can be used as a catalyst in the enzymatic reaction between the macrolide antibiotic and the acylating agent, as well as.

4 "already isolated and disclosed
Position acylase gene (referred to as acyB) (JP-A-2-72)
(See Japanese Patent Publication No. 880), an industrially useful tylosin acylated derivative, 3-O-acetyl-4 ″-.
O-isovaleryl tylosin can be easily produced by fermentative production.

As described above, the macrolide construct 3-position acylase gene (acyA) provided by the present invention.
The DNA fragment containing is widely useful.

<Transformation of Microorganism with Plasmid Incorporating DNA Restriction Fragment Containing acyA Gene>
The plasmid incorporating the acyA gene can be introduced into an appropriate host microorganism depending on the vector plasmid. The host microorganism may be a microorganism capable of producing a macrolide antibiotic or may be a microorganism not producing a macrolide antibiotic. Stoensis (Streptomyces kitasatoensis), Streptomyces narbonencil bal Jiosamyceticus (S. narbonensis varjosamyceticus), Streptomyces hygroscopicus (S. hygroscopicus),
Streptomyces platensis (S. platensis),
S. albiretic
uli), Streptomyces cinerochromogenes (S.
cinerochromogenes), Streptomyces jikartensis (S. djakartensis), Streptomyces
Macrosporeus (S. macrosporeus), Streptomyces tendae (S. tendae), Streptomyces deltae (S. deltae), Streptomyces fladiae (S. fradiae), Streptomyces eurythermas (S) .eurythermus), S. ambofaciens, S. ambofaciens, S. kasugaensis, S. erythreus, S. erythreus, S. erythreus. kanamyceticus) and the like.

However, generally, a macrolide antibiotic-producing bacterium that does not substantially produce the acylating enzyme at the 3-position of the macrolide antibiotic, for example, an anthoramycin-producing bacterium Streptomyces eurythermus ATCC 149.
75, tylosin-producing bacterium Streptomyces fladiae ATCC 19609 and the like are particularly suitable, and particularly the latter Streptomyces fladiae ATCC 196.
09 is preferable.

Transformation of these host microorganisms with a recombinant plasmid carrying the acyA gene can be carried out by a method known per se, for example, the method of Hoppwood [Genetic Manipulation of Streptomyces.
Genetic Manipulation o
f Streptomyces: A Laboratory Manual) 1985,
The John Iness Instate
nnes Institute)].

The transformant thus obtained is cultured in a medium containing a macrolide antibiotic,
Macrolide antibiotics that are acylated at the 3-position can be generated. Alternatively, in the case of a transformant capable of producing a macrolide antibiotic, culturing in a medium in which no macrolide antibiotic is added to the transformant also allows the macrolide antibiotic in which the 3-position is acylated. A substance can be generated.

Further, the transformant is cultivated, and the macrolide antibiotic is acylated in the presence of the cultured cells or a treated product thereof (for example, a cell-free extract obtained by disrupting the cultured cells by sonication). A 3-acylated macrolide antibiotic can also be produced by reacting with an agent, for example, acetyl coenzyme A, propionyl coenzyme A, or a biosynthetic precursor for donating an acyl group, such as leucine.

Thus, according to the present invention, macrolide antibiotics acylated at the 3-position with an alkanoyl group having 1 to 3 carbon atoms such as acetyl and propionyl groups, such as 3-O-acetyltylosin and 3-O. -Propionyl tylosin, 3-O-acetylangoramycin, 3-O
-Propionylangoramycin, 3-O-acetylspiramycin, 3-O-propionylspiramycin and the like can be produced.

Further, a recombinant plasmid in which acyA and the 4 "-position acylase gene (acyB) are simultaneously incorporated as described above, or one in which acyB is incorporated in another plasmid is used. When used simultaneously with a plasmid incorporating acyA, macrolide antibiotics in which the 3 and 4 "positions are simultaneously acylated, such as 3-O-acetyl-4" -isovaleryl tylosin, 3-O-acetyl-.
4 "-n-butyryl tylosin, 3-O-acetyl-
4 "-O-propionyl tylosin, 3-O-acetyl-4" -O-acetyl tylosin, 3-O-propionyl-4 "-O-isovaleryl tylosin and the like can be produced.

<Cultivation of Transformant> A host microorganism transformed with a plasmid containing the DNA fragment containing the acyA gene of the present invention, for example, Streptomyces sp. It can be cultured by the method. Preferred carbon sources added to the culture medium include, for example, molasses, glucose, starch, fats and oils, glycerin, etc., and nitrogen sources include, for example, soybean powder, amino acid mixture, dry yeast and peptones. Nutrient inorganic salts can also be added to the medium, including, for example, the usual salts capable of releasing potassium, sodium, magnesium, calcium, phosphate, chloride and sulphate ions and the like. Essential trace ingredients such as vitamins can also be added if desired. These minor constituents can be supplied in the form of impurities usually associated with the addition of other medium constituents. If necessary, a macrolide antibiotic to be acylated can be added to the medium.

In the case of the transformed Streptomyces microorganism, it should be cultivated at a temperature range of about 20 to 40 ° C. under aerobic conditions in a medium having a pH ranging from about 5 to 9 over a relatively wide range. You can At that time, conditions necessary for maintaining the stability of the plasmid, for example, a drug such as thiostrepton can be added as a selective pressure.

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

[0048]

Example 1 Streptomyces thermotolerance ATCC 11
Preparation of 416 genomic DNA (A) Streptomyces thermotolerance ATC
C 11416 culture 28 in slant medium consisting of glucose 0.4%, yeast extract 0.4%, malt extract 1.0% and agar 1.5%.
One platinum loop of the above strain, which had been cultivated at ℃ for 2 weeks, was used to dissolve soluble starch 2%, soybean powder 2%, yeast extract 0.1%, K ₂ HP.
25 ml of seed mother medium consisting of 0.1% O ₄ and 0.05% MgSO ₄ .7H ₂ O were inoculated. The seed mother medium used was placed in a 250 ml Erlenmeyer flask and sterilized at 120 ° C. for 15 minutes. The inoculated flask was shake-cultured at 28 ° C. for 48 hours to give a seed mother. 28 ml after inoculating 1 ml of seed seed into 25 ml of TSB medium (tryptasease soy broth) *
The cells were cultured for 48 hours.

* TSB medium was prepared at a concentration of 30 g / l.

TSB medium is Difco Laboratories (Dif, Detroit, Michigan, USA).
Co Laboratories).

(B) Preparation of genomic DNA The bacterial cells were collected and washed once with a 10.3% sucrose solution. Next, 25% sucrose / Tris-HCl
(50 mM, pH 8) was added in an amount of 5 ml per 1 g of wet cell weight. After the cells were well dispersed, 0.6 ml of lysozyme [Grade I from Sigma Chemical Co.] solution (10 mg / ml) was added and mixed well. 30 at 30 ° C
Incubate for minutes, followed by 0.6 ml of EDTA solution (0.5 M, pH 8) and 0.1 ml of Pronase E [Sigma.
Chemical Chemicals Co., Ltd.] After adding a solution (10 mg / ml), 30
Incubated at 5 ° C for 5 minutes. 3.6 ml of 3.3% SD
A solution of S [manufactured by Sigma Chemical Co.] was added and incubated at 37 ° C. for 1 hour. 500 g of phenol and 500 g of chloroform were added to Tris-HCl (50 mM, pH 8) / NaCl
6 ml of a phenol layer obtained by mixing 200 ml of (100 mM) / EDTA (5 mM, pH 8) was added and mixed well under mild conditions. The solution is then centrifuged (10,000 rpm, 2
The layers were separated for 0 minutes) to obtain about 7 ml of an aqueous layer. Add 2 ml of chloroform to the aqueous layer, mix well under mild conditions, and then centrifuge (10,000 rpm, 10 minutes) to obtain approximately 7 ml of aqueous layer.
Got 30 μl of liponuclease A type I in the aqueous layer
-Add AS (manufactured by Sigma Chemical Co.) solution (10 mg / ml solution heated at 90 ° C for 10 minutes) and add 37 ° C.
Incubated for 1 hour. After 0.1 volume of sodium acetate solution (3M, pH 4.8) and 1 volume of isopropanol were added and mixed well, the mixture was allowed to stand at room temperature for 10 minutes. The precipitate was collected by centrifugation (6,000 rpm, 10 minutes). Precipitate 5 ml TE buffer [Tris-HCl (10 mM, p
H8) / EDTA (1 mM, pH 8)] and then 0.5
A sodium acetate solution (1 ml) and ethanol (12.1 ml) were added, mixed well, and allowed to stand at -20 ° C overnight. The precipitate was collected by centrifugation (6,000 rpm, 10 minutes) and dried in a desiccator under vacuum. About 25Og of genomic DNA was obtained from 25ml of culture solution.

[0052]

[Example 2] Cloning of gene region related to carbomycin biosynthesis (A) Preparation of cosmid vector pHC79 A single clone of Escherichia coli HB101 [Nippon Gene Co., Ltd., transformation kit] transformed with pHC79 (Boehringer Mannheim) One colony was treated with L broth (1% bactotryptone, 0.5% yeast extract, 0.5% NaC) containing a final concentration of 50 μg / ml ampicillin.
2 ml of pH 7.4) was inoculated and cultured with shaking at 37 ° C. overnight. Add this ampicillin (50 μg / ml) L
Broth (500 ml) was added, and the mixture was again shake-cultured overnight at 37 ° C. After culturing, centrifugation was performed at 5,000 rpm and 4 ° C. for 10 minutes, and the obtained pellets were mixed with 25 mM Tris-HCl pH 8, 10 mM E.
20 ml of solution A consisting of DTA and 50 mM glucose was suspended on ice. Next, 40 mg of lysozyme (Seikagaku Corporation) was dissolved in 0.5 ml of water, and the mixture was gently mixed. This is left at room temperature, and when the cells start to dissolve (usually after 2-5 minutes), 40 ml of freshly prepared and cooled fresh solution B (1% SDS, 0.2N NaOH) is used.
Was quickly added and gently mixed, and the mixture was left again on ice for 5 minutes. Then, 30 ml of chilled solution C (29.3% potassium acetate, 11.5% acetic acid) was added, and the mixture was vigorously shaken and then left on ice for 5 minutes. This is 8,000 rpm, 4
About 90 ml of the supernatant obtained by centrifuging at ℃ for 10 minutes, 0.6
A volume of isopropanol was added, the mixture was allowed to stand at room temperature for 10 minutes, and then centrifuged again at 8,000 rpm and 15 ° C. for 10 minutes to obtain a precipitate. The precipitate was washed once with 50 ml of 70% ethanol and then well dried at room temperature under reduced pressure. The precipitate was then added to 8.2 ml TE (10 mM Tris-HCl, pH 8, 1 mM E).
Dissolved in DTA), 8.4 g cesium chloride and 10 mg
0.2 ml of a / ml ethidium bromide solution was added sequentially. Add this solution to two centrifuge tubes "quick seal".
Dispense to [Bettsmann Co., Ltd.] and use vTi65 rotor [Betsukmann Corp.] at 65,000 rpm for 4 hours or 55,
It was centrifuged at 000 rpm for 15 hours. Thereafter, the band containing the plasmid that appeared in the tube was collected with a 5 ml syringe (Terumo). This solution was extracted 3 to 4 times with an equal amount of n-butanol to remove ethidium bromide, and plasmid DNA was extracted with 3 volumes of 70% ethanol.
The precipitate was washed with 70% ethanol three times, and finally dissolved in 0.5 to 1 ml of TE buffer. By this method, 300 µg of pHC79 plasmid DNA was obtained from 500 ml of L broth.

(B) Construction of S. thermotolerans Gene Library The construction of the library by pHC79 is essentially Ishi-Ho.
by Rowicz, Burke, Nucleic Acids Research, 9: 2987,
(1981) and followed the method taught. Ie pH
C79 50μg each divided into two, one with HindIII and the other with S
It was linearized with alI and dephosphorylated with calf intestinal alkaline phosphatase [Boehringer Mannheim Yamanouchi Co., Ltd.]. Then, both DNAs were digested with BamHI to form left and right arms capable of ligating only one end.
1.5 μg each of the left and right arms and S. thermotolerans
Sau 3AI partial degradation product of genomic DNA (see Example 1) [S. thermotolerans genomic DNA 50 μg was added to Sau 3AI4
Degradation products digested for 2 minutes at 37 ° C in 200 μl Sau 3AI buffer were combined to 4 μg to make 12 μl, and T ₄ DN
0.5 μl (300 μ) of A ligase (Nippon Gene) was added and ligation was carried out at 16 ° C. overnight. After reaction DN
A was concentrated by ethanol precipitation and a portion (equivalent to 4.6 μg) of in vitro packaging kit
Gold (Stratagene) was used for in vitro packaging according to the protocol attached to the kit.
Furthermore, according to the conditions described in the protocol, E. coli HB
As a result of transfecting 101 and examining the titer, 2.8
× 10 ⁵ Colony forming unit (cfu).

(C) Acquisition of cosmid having carbomycin biosynthesis-related gene region Approximately 19,000 colonies of the above gene library were collected from Davis et al., Basic Methods in Molecular Biology (Basic Methods in Molecular B).
(Biology), pages 227-229, 1986, Elsevier Science Publishing Co., In.
c.), 52 Vanderbilt A
venue), New York, New York 1
Colony hybridization was performed according to the method taught in 0017 (New York), USA. DN containing a macrolide 4 "-acylase gene (acyB) nick-translated with [α- ³² P] dCTP [Amashiyam Jaipan Co., Ltd.] as a probe
A part of the A fragment, specifically EcoRI-SacI of the plasmid pY-17ΔEco described in JP-A-2-72880.
A 1 kb fragment was used. For more information on nickel translation, see Molecular Cloning [Molecular Clonin
g, Maniatis, Fritsc
h), Sambrook, Cold Spring Harbor Laboratories
atory), Box100, Cold Spring Harbor, New York] 1
It is described in detail on pages 09-112. As a result, 19 cosmid clones containing a gene region related to carbomycin biosynthesis paired with the probe were obtained. For the purpose of further expanding the cloned region, the plasmid pS was selected from the SE26 strain by the method described in the section A of this Example.
E26 was isolated and screened again from the library using the 1.0 kb BamHI restriction fragment of this cosmid as a probe. As a result, about 20 positive clones including BM73 strain having cosmid pBM73 were obtained.

[0055]

Example 3 Construction of Shear Turkey Smid pIHY-17 (A) Preparation of pIJ922 Streptomyces lividans TK64 / PIJ922 [The Jiyoung Innes Streptomyces culture
The collection (The John Innes StreptomycesCulture C
ollection), Jiyoung Innes Institute (Jo
hn Innes Institute, Colney Lan
e), Norwich NR4 7UH, England (En
gland)] about 10 ⁸ spores of thiopeptin *
YEME + 34% sucrose medium containing 5 μg / ml (yeast extract 0.3%, bactopeptone 0.5%, malt extract 0.3%, glucose 1%, sucrose 3)
4%, after sterilization, 1 ml of 2.5 M MgCl ₂ solution was added) 50
0 ml was inoculated and cultured with shaking at 28 ° C. for 48 hours.

* Tiopeptin (prepared from Fujisawa Pharmaceutical Co., Ltd. by Thiophweed by chloroform extraction) was used as a selective pressure instead of thiostrepton [Pesca and J. W. Boldley, Antibiotics. III (Antibiotics III) 55 pages 1-573,
1975 Springer Fuerlag (Spring-Verla
g)] The cells were collected and washed once with a 10.3% sucrose solution. Then 45 ml of 10.3% sucrose /
Tris-HCl (25 mM, pH 8) / EDTA (21 mM, p
H8) solution and suspended in 5 ml lysozyme solution (10 mg / m
l, dissolved in the same solution used for cell suspension) and 250 μl of ribonuclease type I-AS solution were mixed well and then incubated at 37 ° C. for 30 minutes. Subsequently, 30 ml of 0.3 M NaOH / 2% SDS solution was added and mixed well, and incubated at 55 ° C. for 15 minutes. The same phenol solution as used in Example 1 (B) was added to 2
Add 0 ml and mix well. Centrifuge (15,000 rpm, 1
The layers were separated for 5 minutes) to obtain about 70 ml of an aqueous layer. There 7
ml sodium acetate solution (3M, unadjusted pH) and 70 ml
Isopropanol was added and mixed well, and the mixture was allowed to stand at room temperature for 10 minutes. The precipitate was collected by centrifugation (15,000 rpm, 15 minutes), and 10 ml of TNE buffer [Tris-HC
l (10 mM, pH 8) / EDTA (1 mM, pH 8) / NaCl
(50 mM)], 5 ml of the phenol solution described above was added, and the mixture was stirred well.

Next, the layers were separated by centrifugation (15,000 rpm, 15 minutes) to obtain about 10 ml of the upper layer. 1 ml of sodium acetate (3M, pH 6) and 10 ml of isopropanol were added thereto and mixed well. Centrifuge (1
The precipitate was collected at 5,000 rpm for 15 minutes), washed with 1 ml of ethanol and dried. After dissolving the precipitate in 11.9 ml TE buffer, 12.6 g cesium chloride was added, followed by 0.6 ml ethidium bromide solution (10 mg / ml). After centrifugation at 36,000 rpm for 60 hours, the fraction containing the plasmid band was extracted 5 times with TE buffer and isobutanol saturated with cesium chloride to remove ethidium bromide. After that, put it in the dialysis tube for TE
It was dialyzed against buffer for 24 hours. About 50 μg of plasmid pIJ922 was obtained.

(B) Assembling procedure of pIHY-17 Escherichia coli cosmid pHC79 (for preparation, see Example 2)
2 μg was digested with EcoRI and HindIII and then extracted and purified.
Dissolved in μl TE. On the other hand, pY-17ΔEco (Japanese Patent Laid-Open No. 2-7
(See Japanese Patent No. 2880) 50 μg is similarly added to EcoRI and HindIII
After digestion with TAE buffer-0.8% agarose gel [40 mM Tris HCl, 20 mM sodium acetate, 1 m
Electrophoresed at 6 v / cm with M EDTA, (adjusted to pH 7.2 with acetic acid, 0.8% magalose), stained with ethidium bromide for 4 hours, and then cut out about 6 kb band on the transilluminator. The 6 kb fragment in the gel piece was extracted and purified by GENE CLEAN KIT (Bio101) according to the attached protocol.

0.6 μg of this fragment and the above-mentioned pHC79
EcoRI-HindIII degradation product 0.2 μg (2 μl) in 20 μl 3
00 Yunitsuto T ₄ DNA ligase (Nitsupon Jean)
Incubated at 16 ° C. overnight. 5 μl of this reaction solution
Was added to 150 μl of Escherichia coli HB101 competent cell for transformation.

For details of transformation, see Davis et al., Basic Methods in Molecular Biology 70-90.
The method described on the page was followed.

Transformed strain obtained (acipicillin resistance)
From 6 strains on a small scale,
The presence or absence of the 6 kb fragment was examined from the migration pattern of the EcoRI-HindIII degradation product. As a result, 3 strains had the desired 6 kb fragment. Example 2 from one of these
A plasmid was prepared by the method shown in (A).

5 μg of this plasmid was linearized with EcoRI,
It was dephosphorylated with calf intestine-derived alkaline phosphatase [Boehringer Mannheim Co., Ltd.]. This DNA 1.2
5 μg and pIJ92 prepared in (A) of this example
2 μl of EcoRI linearized DNA of 2 was reacted with 200 units of T ₄ DNA ligase (Nippon Gene) in 7 μl at 16 ° C. for 4 hours. Using 4 μl of the reaction solution, in vitro packaging was performed with a Gigapack Gold Kit (Stratagene), and Escherichia coli HB101 was transduced.

Among the thus obtained HB101 transduced strains, the plasmids of 6 strains were examined on a small scale, and it was found that 4 strains contained the desired plasmid pIHY-17 (see FIG. 2). A large amount of plasmid was prepared from one of these strains in the same manner as in Example 2 (A), and as a result, 1.65 mg of plasmid DNA was obtained from 1000 ml of L broth.

[0064]

Example 4 Acquisition of p53A (A) Incorporation of a DNA fragment of a carboxycin biosynthesis-related gene region into pIHY-17 25 μg of pIHY-17 prepared in Example 3 above was prepared according to the method described in Example 2. Hind II with one divided into two
I and the rest were digested with EcoRI and dephosphorylated with alkaline phosphatase.

Next, these were cleaved with XhoI to give T of each degradation product.
30 kb HindII from AE-0.6% agarose electrophoresis
The I-XhoI fragment and the 11 kb EcoRI-Xho fragment were separated and purified [GENE CLEAN KIT (Bio101)], and 1 μg of each fragment and a cosmid clone containing the carbomycin biosynthesis gene region obtained by the method described in Example 2 were obtained. Completely digested product of XhoI (such as pSE26 and pBM73) 1μ
g and 2 μg of incomplete degradation product of the same SalI were mixed, and mixed in 25 μl
50 Yunitsuto T ₄ DNA ligase (Nitsupon Gene), overnight Inkiyubeto at 16 ° C..

After the reaction, the DNA was precipitated and 5 μl of T was added again.
E-dissolved in buffer. 2.5 μl of this solution
In vitro packaging [Gigapack Gold Kit (Stratagene)] Then, E. coli HB101 was transduced. As a result, about 2,500 Escherichia coli clones thus obtained were collected by suspending them from the plate in 10 ml of L broth, and the mixed plasmid was extracted using the method described in Example 2 on a small scale to obtain 20 μl of TE. This was used as a solution and used for the transformation described below.

(B) About 10 spores of transformed Streptomyces lividans TK24
^{Eight of the} same 25 ml YEME + used in Example 3
34% sucrose medium (5 mM MgCl ₂ and 0.
Inoculate a spring-loaded 250 ml Erlenmeyer flask containing 5% glycine and no thiopeptin),
The culture was performed at 28 ° C. for 32 hours with shaking.

Cells were collected by centrifugation and 10 ml of 10.3%
After washing with sucrose, it was well suspended in 4 ml of P medium containing 1 mg / ml of lysozyme, and incubated at 30 ° C. for 45 minutes. P medium is 10.3 g of sucrose, K
_{2 SO 4 0.025g, MgCl 2 ·} 6H 2 O 0.202g,
Trace metal solution * 0.2 ml to 80 ml with deionized water 12
After sterilizing at 0 ℃ for 15 minutes, sterilized separately 0.5% K
H ₂ PO ₄ 1 ml, 3.68% CaCl ₂ · 2H ₂ O 10 m
l, TES [2-([tris- (hydroxymethyl) methyl] amino) ethanesulfonic acid] buffer (0.2
It was prepared by adding 10 ml of 5M, pH 7.2).

* A trace metal solution contains the following salts in 1 liter: ZnCl ₂ 40 mg, FeCl ₃ .6H ₂ O 200 m
g, CuCl ₂ .2H ₂ O 10 mg, MnCl ₂ .4H ₂ O 10 m
g, Na ₂ B ₄ O ₇ · 10H ₂ O 10 mg, (NH ₄ ) ₆ Mo ₇ O
After ₂₄ · 4H ₂ O 10mg Inkiyubeto to remove hyphae are Zantsu cotton filtration. The protoplasts were collected by centrifugation (3,000 rpm, 10 minutes) and resuspended in P medium at 3-4 × 10 ⁷ cells / 100 μl. DNA of the above-mentioned plasmid
10 μl of the solution was added, and transformation was performed in the presence of 25% PEG 1000 [manufactured by BDH Chemical (Chemical)].

The protoplasts transformed with P medium were washed and then resuspended in 1 ml of P medium. Its 0.1m
l was inoculated onto R2YE agar plate and incubated at 28 ° C. for 16 hours. After culturing, the final concentration of thiopeptin is 50 μg
A soft agar medium containing thiopeptin was overlaid so that the concentration became / ml, and the cells were further cultured at 28 ° C. for 72 hours. R ₂ YE plate is 10.3 g of sucrose, K ₂ SO ₄ 0.02
5 g, MgCl ₂ .6H ₂ O 1.012 g, glucose 1 g,
Deifukokazamino acid 0.01 g, and Bacto agar 2.2g were dissolved in distilled water 80 ml, 120 ° C., was sterilized for 15 minutes, each sterile aforementioned trace metal solution 0.2 ml, 0.5% KH ₂
PO ₄ 1 ml, 3.68% CaCl ₂ .2H ₂ O 8 ml, 20%
L-proline 1.5 ml, TES buffer (0.25 M, p
It was prepared by adding 10 ml of H7.4), 0.5 ml of 1N NaOH and 5 ml of 10% yeast extract and dispensing 20 ml of the mixture into a plastic sticky dish having a diameter of 9 cm. Then, it was dried in a clean bench for about 2 hours. On the other hand, the soft agar medium used was 8 g of Nutrient broth and 3 g of Bacto agar made up to 1 liter with distilled water and sterilized at 120 ° C. for 15 minutes.

(C) Isolation of Transformant Having Acylation Activity Approximately 200 strains out of about 1,000 transformant strains obtained were each subjected to glucose 1%, yeast extract 0.5%, malt extract 1%, pH 7.2, Bact agar 1.5% and Thiopeptin 5
It was inoculated into an agar medium containing 0 μg / ml and cultured at 28 ° C. for 4 days.

Leucomycin A ₁ final concentration is 500 μg
/ Ml was added to a soft agar medium containing leucomycin A ₁ and the culture was continued for 48 hours. Punch the growing colony center with a cork borer with a diameter of about 6 mm,
Put the agar piece on a silica gel TLC plate [Whatman (What
man), LK6DF] and air dried.

Plates were n-hexane: acetone: benzene: methanol: ethyl acetate = 30: 10: 25:
After developing with a developing solvent consisting of 8:20, the product spot was examined by sulfuric acid color development, and an attempt was made to detect leucomycin A ₁ which was acylated at the 3-position. About 200 strains of Streptomyces lividans 53A strain (hereinafter 53
Referred to as A strain) was found to produce a leucomycin A ₃ is leucomycin A ₁ which is acylated. 5
Leucomycin A ₃ produced by 3A strain is TLC, HP
It was identified as leucomycin A ₃ from the analysis results of LC, UV spectrum, NMR spectrum, antibacterial spectrum and the like.

This strain has been deposited as Micro Engineering Research Article No. 2893 (FERM BP-2893) at the Institute for Microbial Technology, Ministry of International Trade and Industry, 1-13 East, Tsukuba City, Ibaraki Prefecture.

A plasmid was isolated from strain 53A substantially according to the method described in Example 3 to obtain plasmid p53.
I got A. About 3.2 kilobases of foreign DNA had been inserted into this plasmid. FIG. 3 shows the restriction enzyme cleavage site and a function map. For the sake of convenience, not all of the cutting positions of the restriction enzymes are shown, but they are shown as far as they can be characterized.

[0076]

Example 5 (A) Transformed with 53A strain and p53A
Confirmation of 3-position acylation activity of S. lividans 53A strain or pA53 retransformed strain was treated with 1% glucose, 0.5% bacto yeast extract, 1% malto extract, 1.5% bacto agar and NaO.
After adding H to adjust the pH to 7.2, the cells were inoculated into a circle with a diameter of about 2 cm on an agar medium containing 5 μg / ml of thiopeptin.
After 4 days at 0 ° C., 2 ml of soft agar containing leucomycin A ₁ was overlaid so that the final concentration was 500 μg / ml.

After 2 days at 28 ° C., the central part of the colony was extracted with a cork borer, and the agar pieces were finely crushed in an Eppendorf tube using a plastic stick. To this crushed product, 50 μl of toluene was added and vigorously extracted by stirring. The upper toluene layer after centrifugation (16,000 rpm, 5 minutes) was taken and subjected to thin layer chromatography. The thin layer plate used was Art 5715 manufactured by Merck, and the developing solvent described in Example 4 was used.

After development, the plate is dried and 10% H ₂ S
The spots were confirmed by heating and coloring at 105 ° C. for 5 minutes after dipping in O ₄ . Leucomycin A ₁ has an Rf (relative mobility) value of 0.49. From the toluene extract of the 53A strain or the retransformed strain, in addition to this unconverted leucomycin A ₁ , leucomycin A ₃ with Rf = 0.60 was obtained.
(OH-acetylated 3-position of leucomycin A ₁ ) was observed. In the control strain not transformed with the plasmid p53A (for example, the transformant of the vector pIHY-17), the production of the 3-acylated form of leucomycin A ₁ was not observed.

(B) Confirmation of leucomycin A ₃ S. lividans 53A strain was treated with 1 L of TSB medium containing 5 μg / ml of thiopeptin (triptycase soy broth).
At 28 ° C with shaking culture (500 ml containing 100 ml of medium)
10 ml flasks. Each flask was inoculated with 53A strain-platinum loop) and leucomycin A ₁ was added on the 3rd day (final concentration 200 μg / ml). On day 4, the culture was extracted with 500 ml of ethyl acetate at pH 9.0 and then 0.01M KH _2.
After being dissolved in 500 ml of PO ₄ solution (adjusted to pH 3.0 with HCl), NaHCO ₃ was added again to adjust the pH to 9.0, and the mixture was extracted with 500 ml of ethyl acetate. The extract was evaporated to dryness in a vacuum concentrator and then dissolved in 2 ml of methanol. The whole amount of this solution was injected into HPLC. The condition of HPLC is that column YMC-Pack S-343I-15 ODS (Yamamura Kagaku) is used, the flow rate is 5 ml / min at room temperature, the mobile phase is 0.1 M NaH ₂ PO ₄ ,
0.3M NaClO ₄ adjusted to pH 2.5 with phosphoric acid 1
It is assumed that 2 volumes of Meta Rule are added to the volume.

The specific peak (retention time 80 minutes) detected at UV230 nm was collected. The methanol in the solution was removed by a vacuum concentrator, adjusted to pH 9.0 with NaOH, and extracted with ethyl acetate. The extract was evaporated to dryness in a vacuum concentrator, dissolved in chloroform, and evaporated to dryness again to obtain 2 mg of a white powder. The results of examining the physicochemical properties of this substance are shown below.

[0081] · TLC [Merck (Merck) Art5715 used] specimen Rf value leucomycin _{A 1} 0.49 leucomycin _{A 3} 0.60 S. lividans 53A product 0.60 (developing solution: n-hexane: acetone: benzene: methanol: ethyl acetate = 30: 10: 25: 8: 20) ・¹ H-NMR (400MHz, CDCl ₃ , TDS standard) 0.97 (d, 6H, J = 6.6Hz, 4 "-O-COCH ₂ CH (C H ₃ ) ₂ 2.26 (s, 3H, 3-O-COC _{H 3) 2.51 (s, 6H} , 3'-N (C H 3) 2) 3.52 (s, 3H, 4-OC H 3) 4.42 (d, 1H, J = 7.3Hz, H-1 ') 4.61 ( d, 1H, J = 10.3Hz, H-4 ") 5.06 (d, 1H, J = 2.9Hz, H-1") 5.11 (d, 1H, J = 11.0Hz, H-3) 5.60 (dd, 1H , J = 15.4, 9.5Hz, H-10) 6.06 (dd, 1H, J = 11.0, 14.7Hz, H-12) 6.58 (dd, 1H, J = 11.0, 15.4Hz, H-11) 9.62 (s, 1H, H-18) shows a ¹ H-NMR spectrum diagram in Figure 4.

The above results are based on literature values [S. Omura et al.,
The Journal of Antibiotics (The Jo
urnal of Antibiotics), 23, 511-513,
(1970)] and was determined to be leucomycin A ₃ .

[0083]

Example 6 Construction of pMAA2 and pMAA3 50 μg of p53A (see FIG. 3) was digested with SphI and TA
Electrophoresis on E-buffer-0.8% agarose gel, 2k
The two types of bands existing in the vicinity of b were extracted and purified as a mixture with Gene Clean Kit (manufactured by Bio101). 0.6 μg of these fragment mixtures and E. coli vector pUC
300 units of T ₄ DNA ligase (Nippon Gene) was added to 0.2 μg of 19 SphI linearized DNA, and the mixture was incubated at 16 ° C. for 4 hours.
Incubated for an hour. E. coli J with 10 μl of this reaction solution
M103 competent cells were transformed with ampicillin-
Xgal medium (Bactotryptone 1%, Bacto yeast extract 0.5%, NaCl 1%, Bacto agarto 1.5g pH 7.4, ampicillin 50 after autoclaving
μg / ml, 0.5 mM IPTG, Xgal 0.01% addition)
Then, six white colonies, that is, strains in which the inactivation of the lacZ gene of the vector pUC19 had occurred were picked up, and plasmids were extracted on a small scale.

Then, it was confirmed by the electrophoresis patterns of these plasmids by restriction enzymes which of the two kinds of fragments was inserted. That is, the plasmid was reacted with BglII, and it was examined which fragment it had depending on whether it was linearized or intact. As a result, the former plasmid (Bg
There were 2 strains with one lII site) and 4 strains with the latter plasmid (no BglII site in the insert). One strain was selected from the former, this plasmid was named pMAA2, and one strain was selected from the latter, and this plasmid was named pMAA3 (see FIG. 5).

PMAA2 and pMAA3 were prepared in large quantities according to Example 2. Further, the restriction sites of the inserted fragments of these plasmids were determined in detail to create a restriction enzyme map of the DNA fragment containing the acyA gene (see FIG. 1).

[0086]

Example 7 Nucleotide Sequence Determination of acyA The 3-acylating enzyme gene acyA can be easily excised from the plasmid p53A with the restriction enzymes BamHI and SalI, and this sequence (1810 bp) is a commercially available sequence kit. Dear The Sea Kenace Version 2.
0 (United States Biochemical Corporation / Cleavela
nd, Ohio, USA). The sequence is 6th-9th
Shown in the figure.

The acyA coding region was found at positions 209 to 1375. There is one ribosome binding site consisting of AAGGA near the upstream of the initiation codon, ATG, on the 5 ′ side.
Seen at the 94th to 198th positions and the 201st to 205th positions, and in the upstream region, there are a promoter region, that is, TGCCG (120th to 125th) which is the -35 region and CAGGAT (143th to 148th) which is the -10 region. Was there. The relationship between the base sequence of acyA and the amino acid sequence encoded thereby is shown in FIGS.

[0088]

[Example 8] Production of acylated tylosin (A) Preparation of pAB5 2 μg of recombinant plasmid pMAB1 (see Japanese Patent Laid-Open No. 2-72880) carrying the macrolide 4 "-position acylating enzyme gene acyB (18 µl, 10 times TE buffer) Concentration of XbaI buffer (NaCl 500 mM, Tris-HCl (pH 7.9) 200 mM, MgCl ₂ 100
mM, 2-mercaptoethanol 100 mM) 2 μl,
Add 1 μl (15 units) of the restriction enzyme XbaI,
The reaction was carried out at 37 ° C for 2 hours. This DNA solution was saturated with an equal amount of TE buffer, extracted with a phenol / chloroform mixed solution (phenol: chloroform: isoamyl alcohol = 25: 25: 1, 0.1% quinolinol TE buffer saturated solution), and then extracted with 3M sodium acetate. DNA was precipitated by adding 1/10 volume of the solution (pH 5.2) and 3 times volume of ethanol. This DNA was washed with 70% ethanol, dissolved in 8 μl of TE, and added with 10 times concentration of exonuclease III buffer (Tris.HCl (pH
8.0) 500 mM, MgCl ₂ 50 mM, 2-mercaptoethanol 100 mM) 1 μl and exonuclease III [Nippon Gene Co., Ltd.] 1 μl (50 unit
s) was added, and the mixture was reacted at 30 ° C for 1 minute, then immediately added with H ₂ O.
24 μl and 10 times concentrated nuclease S1 buffer (sodium acetate (pH 4.5) 330 mM, NaCl
500 mM, ZnSO ₄ 0.3 mM) ₄ μl and nuclease S1 [Boehringer Mannheim Yamanouchi Co., Ltd.] 2 μ
l (20 units) was added and reacted at room temperature for 15 minutes. After the reaction, the DNA was purified by phenol, chloroform extraction and ethanol precipitation in the same manner as described above, and TE23
It was dissolved in μl. To this solution was added 10 times M buffer (NaCl 500 mM, Tris-HCl (pH 7.5)).
3 mM of 100 mM, MgCl ₂ 100 mM, dithiothreitol 10 mM and dNTP mixed solution (dGTP, dCT)
P, dATP, dTTP 0.5 mM each mixed solution) 3
After adding μl, DNA polymerase I [large fragmen
t, Takara Shuzo Co., Ltd.], and add 1 μl (4 units) at 37 ℃
After reacting for minutes, the DNA ends were blunted. After purification by phenol, chloroform, and ethanol precipitation, the obtained DNA was dissolved in 10 μl of TE buffer to obtain a vector solution. On the other hand, a 1.8 kb DNA fragment containing acyA obtained by digesting 100 μg of the recombinant plasmid p53A obtained in Example 4 with the restriction enzymes Sail and BamHI was used to generate a gene clean gene (Gene clean (Bio10
1)] and separated and purified to obtain about 4 μg of the target fragment. This DNA fragment was blunt-ended under the same conditions as described above, purified by phenol-chloroform extraction and ethanol precipitation, and then TE solution (0.2 μg / 0.2 μg /
μl). Next, 2.5 μl (0.5 μg) of this solution, 2.5 μl (0.5 μg) of the vector solution described above, and purified water 13
μl, 10 times concentration of ligation buffer (Tris · HCl 500 mM (pH 7.9), MgCl ₂ 100
mM, dithiothreitol 200 mM, ATP 100 m
M) 2 μl and T ₄ DNA ligase [Nippon Gene Co., Ltd.] 1 μl (300 units) were sequentially added, and the mixture was added at 16 ° C.
The reaction was carried out for 15 hours. Use 2 μl of this reaction mixture to
00 μl of E. E. coli JM103 competent cells were transformed. This method is the method of Maniatis et al.
(Molecular Cloning, Cold Spring Harbor, Laboratory (Cold Spring
Harbor, Laboratory). The plasmid possessed by the transformant thus obtained was extracted and examined by agarose electrophoresis. As a result, a plasmid pAB5 having an insert fragment of about 1.8 kb having the structure shown in FIG. 17 was obtained.

(B) 10 μg of pAB10 and pAB10ΔB1 plasmid pAB5 were completely digested with the restriction enzyme SacI, and then phenol / chloroform extraction and ethanol precipitation were carried out to purify the DNA fragment. Then, of the fragments obtained by partial digestion with the restriction enzyme PstI, only the DNA fragment derived from pUC18 was excised, and the resulting DNA fragment of about 9.2 kb was separated by 0.8% agarose gel electrophoresis. , Gene Clean (Gene1
01) was used for recovery and purification. This DNA fragment is TE
It is dissolved in 22 μl of buffer and 10 times concentration of T ₄ polymerase buffer (Tris-HCl (pH 7.4) 700).
mM, MgCl ₂ , 100 mM, dithiothreitol 5
0 mM) 3 μl and dNTP mixed solution (dGTP, dCTP,
After adding 3 μl of 0.5 mM each of dATP and dTTP mixed solution, T ₄ DNA polymerase [Toyobo Co., Ltd.] was added to 2
μl (4.8 units) was added and reacted at 37 ° C. for 30 minutes to blunt the DNA ends. After extraction with phenol / chloroform and purification by ethanol precipitation, the product was dissolved in 45 μl of TE buffer, added with 5 μl of 10-fold concentration ligation buffer and T ₄ DNA ligase [Nippon Gene Co., Ltd.] 1 μl (300 units), and kept at 16 ° C. After 15 hours of reaction, self-cyclization was performed. 20 μl after reaction
Streptomyces lividans (Str
Transformation of protoplasts of eptomyces lividans) TK25 was performed. From among the obtained transformants, pUC18
Plasmids in which a portion was completely deleted were selected based on differences in electrophoretic patterns, and among these, pAB10 having the structure shown in FIG. 18 and pAB1 having the structure shown in FIG.
0 ΔB1 was obtained. Both plasmids are p
It is the same in that the UC18 region is deleted, but in the latter (pAB10ΔB1), as a result of the side reaction of the enzyme, the deletion further extends to the 4 "-position acylase gene acyB region, and thus the 4" -position. Unable to produce acylating enzyme.

(C) Production of 3-O-Acetyltylosin One platinum loop amount of a slant culture of Streptomyces fradeier ATCC 19609 was treated with GPY medium (glucose 1.0%, polypeptone 0.4%, yeast extract 0.4). %,
_{MgSO 4 · 7H 2 O 0.05%} . K ₂ HPO ₄ 0.1%
And glycine 0.05%, pH 7.2) 20 ml was added 2
A 50 ml Erlenmeyer flask was inoculated and shake-cultured at 28 ° C. for 48 hours. 0.5 ml of this culture solution was used for YE shown in Example 3.
ME + 15% sucrose medium (5 mM MgCl ₂
Inoculate a 250 ml Erlenmeyer flask with spring containing 25 ml (including 0.5% glycine), and incubate at 28 ° C for 4
It was shake-cultured for 0 hours. Centrifuge (3,000 rpm, 15
The cells were collected by 10 minutes), washed with 10 ml of 10.3% sucrose and then well suspended in 4 ml of P medium containing 1 mg / ml of lysozyme, and reacted at 30 ° C. for 1 hour. The composition of P medium is as shown in Example 4. After the reaction, the remaining hyphae were removed by cotton filtration, and the protoplasts were collected by centrifugation (3,000 rpm, 10 minutes) and suspended in P medium at 3 × 10 ⁹ cells / 100 μl. The above-mentioned pAB10ΔB1 solution (15 μg /
10 μl) 10 μl was added, and 25% PEG1000 [BD
H-Chemical (manufactured by Chemical). The transformed protoplasts were washed with P medium and then resuspended in 1 ml of P medium. R of 0.1 ml
3 medium (13.5 g of sucrose, KCl 0.05
%, Glucose 1.0 g, polypeptone 0.4 g, yeast extract 0.4 g, TES buffer 0.573 g, 10% M
6.1 ml of gCl ₂ solution, 2.0 g of Bacto agar and 100 ml of distilled water; KH ₂ PO ₄ (0.5) after autoclave sterilization
%) 1.0 ml, CaCl ₂ (5M) 0.3 ml and NaOH
(1N, add 1.8 ml) and inoculate on 1
Cultured for 6 hours. After culturing, the final concentration of thiopeptin is 5μ
The soft agar medium containing thiopeptin was overlaid so that the concentration was g / ml or 25 μg / ml, and the cells were further cultured at 28 ° C. for 7 days. Soft agar is Nutrien broth
1 liter of distilled water was added to 8 g of t broth) and 3 g of Bact agar, and autoclave sterilization was performed at 120 ° C. for 15 minutes.

A large number of colonies appeared on the medium containing 5 μg / ml of thiopeptin, but few resistant colonies appeared on 25 μg / ml. One strain showing resistance to 25 μg / ml was designated as Streptomyces fragier AB10ΔB1 strain (hereinafter referred to as AB10ΔB1 strain), and a production test of acylated tylosin was conducted.

Production medium (soluble starch 0.5%, glucose 0.5%, yeast extract 0.5%, malt extract 1.
One platinum loop of AB10ΔB1 strain was inoculated into a 250 ml Erlenmeyer flask containing a coil containing 0% and 50 ml of thiopebutin (50 μg / ml), and the mixture was shake-cultured at 28 ° C. for 21 days, and 1 ml was taken to obtain 0.2 g of K ₂ HPO _4. And 0.2 ml of toluene was added,
Extracted with vigorous stirring. The upper toluene layer was removed by centrifugation (3,000 rpm, 10 minutes), and thin layer chromatography was performed. The thin layer plate used was [Merck] Art 5715, and the developing solvent was 75 m of ethyl acetate.
l: diethylamine 1.5 ml: water 1.5 ml: methanol 0.
75 ml was used. After drying this plate, the wavelength is 280 nm.
At Chromatoscanner (Shimadzu CS-93
0), further 10% H ₂ SO ₄ immersion, and then 10
The position was confirmed by heating and coloring at 5 ° C. for 5 minutes.
The tylosin Rf (relative mobility) value was 0.62, and the product of the transformant contained Rf =
0.68 3-acetyltylosin was found.

On the other hand, production of 3-acetyltylosin was not observed in the control strain which was not transformed with the plasmid pAB10ΔB1.

(D) Confirmation of 3-O-acetyltylosin From the culture solution of 1 liter of the above-mentioned production medium (20 main cultures containing 50 ml), extraction operation was carried out with ethyl acetate at pH 8.5 to obtain acidic water (0. It was dissolved in 200 ml of 01N HCl, adjusted to pH 8.5 again, and extracted with 50 ml of ethyl acetate. This extract was concentrated to 10 ml and thin layer plate (Merck [Mec [Me
2 pieces of each 2 ml were spotted on 5 sheets of Art 5715 manufactured by rck] and developed with the above-mentioned developing solvent. Air-dried Rf =
The silica gel at the spot of 0.68 was scraped off and extracted with 10 ml of acetone. The extract was evaporated to dryness in a vacuum evaporator and a white powder (20 mg) was obtained. The results of examining the physicochemical properties of this substance are shown below.

Molecular weight: 958 (dehydrated ion peak 941 was obtained by chemical impact mass spectrometry) ¹ H-NMR (δ, ppm): 1.8 (12-CH ₃ ), 2.
_{14 (3-OCOC H 3)} , 2.5 (N (CH 3) 2),
_{3.5 (3 "-OCH 3),} 3.6 (2"'- OCH 3),
4.15 (H1 '), 4.55 (H1 "'), 4.60 (H
4 "), 5.05 (H1"), 5.95 (H12), 6.
25 (H10), 7.3 (H11), 9.7 (CHO).

FIG. 20 shows the 1H-NMR diagram.

The above results are based on literature values [US Pat.
2,473 and The Journal of Antibiotics, 33
Vol. 1300-1308, 1980]
It was determined to be -O-acetyltylosin.

(E) Transformation with pAB10 One platinum loop of a slant culture of Streptomyces fradeier ATCC19609 was cultured in the same manner as in the above paragraph,
Protoplast formation was performed using lysozyme. As a plasmid, pIJ702 (from Streptomyces lividans ATCC 35287, the method of Hopwood) [Genetic Manipulation of Streptomyces
ces): A Laboratory Manu
al), John Innes
Foundation, Norwich, UK, 1985
Was prepared according to the above procedure), and the resulting transformant was again protoplasted and regenerated by the same method as described above to obtain a strain lacking pIJ702 (designated S. fradiae C-1).

The S. fradiae C-1 thus obtained was cultured and protoplasted by the same method as described above, 10 μl of the above plasmid pAB10 solution (15 μg / 10 μl) was added, and 25% PEG1000 [BDH Chemical (Chemical) was added.
Transformation was carried out in the presence of The transformed protoplasts were washed with P medium and then resuspended in 1 ml of P medium. 0.1 ml of it was added to R3 medium [Sucrose 13.5 g, KCl 0.05%, glucose 1.0 g,
Polypeptone 0.4 g, yeast extract 0.4 g, TES buffer 0.573 g, 10% MgCl ₂ solution 6.1 ml, Bact agar 2.0 g and distilled water 100 ml; KH ₂ PO ₄ (0.5%) after autoclave sterilization. 1.0 ml, CaCl ₂
(5M) 0.3 ml and NaOH (1N) 1.8 ml are added], and the mixture is incubated at 28 ° C. for 16 hours. After culturing,
Final concentration of thiopeptin is 5μg / ml or 25μg / ml
Then, a soft agar medium containing thiopeptin was overlaid so that it was cultured at 28 ° C. for 7 days. Soft agar medium was 8 g of Nutrient broth and Bact
1 l of distilled water was added to 3 g of agar, and autoclave sterilization was performed at 120 ° C. for 15 minutes.

A large number of colonies appeared on the medium containing 5 μg / ml of thiopeptin, but few colonies were resistant on 25 μg / ml. One strain showing resistance to 25 μg / ml was designated as Streptomyces fragier AB10 strain (hereinafter referred to as AB10 strain), and a production test of acylated tylosin was conducted.

Production medium (soluble starch 0.5%, glucose 0.5%, yeast extract 0.5%, malt extract 1.
One platinum loop of AB10 strain was inoculated into a 250 ml Erlenmeyer flask containing a coil containing 0% and 50 ml of thiopeptin (5 μg / ml) and cultured at 28 ° C. for 7 days with shaking.
₂ HPO _{4 (} 0.2 g) and toluene (0.2 ml) were added, and the mixture was vigorously stirred and extracted. The upper toluene layer was removed by centrifugation (3,000 rpm, 10 minutes), and thin layer chromatography was performed. Thin layer plate [Merck]
Using Art5715, the developing solvent is ethyl acetate 75 ml:
Diethylamine 1.5 ml: Water 1.5 ml: Methanol 0.7
5 ml was used. Chromatoscanner (CS-930 manufactured by Shimadzu) at a wavelength of 280 nm after drying this plate
Analysis by 10% H ₂ SO ₄ immersion at 105 ℃
The position was confirmed by heating and coloring for 5 minutes. Tylosin has an Rf (relative mobility) value of 0.62, and the product produced by the transformant was produced in addition to tylosin, 3-O-acetyl-4 ″ -O-isovaleryl tylosin having Rf = 0.82. This strain, S. fradiae AB10, was found at the Institute of Microbiology and Industrial Technology, Ministry of International Trade and Industry, Ministry of International Trade and Industry, 1st-3rd East, Tsukuba City, Ibaraki Prefecture.
It has been deposited as M BP-3212).

On the other hand, in the control strain which was not transformed with the plasmid pAB10, 3-O-acetyl-4 "-O-.
No production of isovaleryl tylosin was found.

G) Confirmation of 3-O-acetyl-4 ″ -O-isovaleryl tylosin The above-mentioned production culture medium 1 l culture medium (50 ml medium 20)
From the main culture), extraction operation was performed with ethyl acetate at pH 8.5. The solution was dissolved in 200 ml of acidic water (0.01 NHCl), adjusted to pH 8.5 again, and extracted with 50 ml of ethyl acetate. The extract was concentrated to 10 ml and thin layer plate (Merck [M
2 pieces of 2 ml each were spotted on 5 sheets of Art 5715 manufactured by Erck] and developed with the above-mentioned developing solvent. After air-drying, scrape off the silica gel from the spot of Rf = 0.82,
It was extracted with 10 ml of acetone. The extract was evaporated to dryness in a vacuum evaporation concentrator to obtain 5 mg of white powder. The results of examining the physicochemical properties of this substance are shown below.

Molecular weight: 1042 (dehydrated ion peak 1025 was obtained by chemical impact mass spectrometry) ^{1 H-NMR (δ, ppm} ) 0.97 (OCCH 2 CH (C H 3) 2), 1.8 (12-
CH ₃ ), 2.14 (3-OCOC H ₃ ), 2.28 (OC
_{C H 2 CH (CH 3)} 2), 2.5 (N (CH 3) 2), 3.
_{5 (3 "-OCH 3),} 3.6 (2 '" - OCH 3), 4.1
5 (H1 '), 4.55 (H1'"), 4.60 (H
4 "), 5.05 (H1"), 5.95 (H12), 6.
25 (H10), 7.3 (H11), 9.7 (CHO), and FIG. 21 shows the ¹ H-NMR diagram.

The above results are confirmed by Okamoto et al. [(R. Okamot
o et al. ): The Journal of Antiques (The Journal of Ant)
ibioticus), 33 volumes, 1300-1308
Page, 1980)] and was determined to be 3-O-acetyl-4 "-O-isovaleryl tylosin.

[Brief description of drawings]

1 (A) and (B) are restriction enzyme maps of a DNA fragment containing the gene acyA of the present invention,

FIG. 2 is a restriction enzyme cleavage point and function map of plasmid pIHY-17,

FIG. 3 is a restriction map and a functional map of plasmid p53A,

FIG. 4 is a ¹ H-NMR chart of leucomycin A ₃ ,

FIG. 5 is a restriction enzyme cleavage point and function map of plasmids pMAA2 and pMAA3,

FIG. 6 is a view showing a part of the base sequence of acyA,

FIG. 7 is a diagram showing a part of the base sequence of acyA continued from FIG. 6,

FIG. 8 is a diagram showing a part of the base sequence of acyA continued from FIG. 7,

FIG. 9 is a diagram showing a part of the base sequence of acyA continued from FIG.

FIG. 10 is a diagram showing a part of an amino acid sequence corresponding to the base sequence of acyA,

FIG. 11 is a diagram showing a part of an amino acid sequence corresponding to the base sequence of acyA continued from FIG. 10,

FIG. 12 is a diagram showing a part of an amino acid sequence corresponding to the base sequence of acyA continued from FIG.

FIG. 13 is a view showing a part of an amino acid sequence corresponding to the base sequence of acyA continued from FIG. 12,

FIG. 14 is a diagram showing a part of an amino acid sequence corresponding to the base sequence of acyA continued from FIG.

FIG. 15 is a diagram showing a part of the amino acid sequence corresponding to the base sequence of acyA continued from FIG.

FIG. 16 is a diagram showing a part of an amino acid sequence corresponding to the base sequence of acyA continued from FIG.

FIG. 17 is a restriction enzyme cleavage point and function map of plasmid pAB5,

FIG. 18 is a restriction enzyme cleavage point and function map of plasmid pAB10,

FIG. 19 is a restriction enzyme cleavage point and function map of plasmid pAB10ΔB1,

FIG. 20: ¹ H-NMR of 3-O-acetyltylosin
It ’s a chart,

FIG. 21 is a ¹ H-NMR chart of 3-O-acetyl-4 ″ -O-isovaleryl tylosin.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] May 8, 1991

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0083

[Correction method] Change

[Correction content]

[0083]

Example 6 Construction of pMAA2 and pMAA3 50 μg of p53A (see FIG. 3) was digested with SphI,
It was electrophoresed on a TAE buffer-0.8% agarose gel, and two types of bands existing in the vicinity of 2 kb were extracted and purified as a mixture with a Gene Clean Kit (manufactured by Bio101). 0.6 μg of a mixture of these fragments and 0.2 of SphI linearized DNA of E. coli vector pUC19
Dissolve μg in TE18μl, and use 10x ligation
2 μl of buffer and 300 units of T ₄ DNA ligase (Nippon Gene) were added and incubated at 16 ° C. for 4 hours. Escherichia coli JM103 competent cells were transformed with 10 μl of this reaction solution to give ampicillin-Xgal.
Medium (Bacto tryptone 1%, Bacto yeast extract 0.5%, NaCl 1%, Bacto agarto 1.
5g pH7.4, ampicillin 50μ after autoclaving
g / ml, 0.5 mM IPTG, Xgal 0.01%
Added), white colonies, ie vector pUC1
Six strains with insertion inactivation in the lacZ gene of 9 were picked up and the plasmid was extracted on a small scale.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0088

[Correction method] Change

[Correction content]

[0088]

Example 8 Production of acylated tylosin (A) Preparation of pAB5 2 μg of recombinant plasmid pMAB1 (see Japanese Patent Laid-Open No. 2-72880) carrying the macrolide 4 ”-position acylating enzyme gene acyB (18 μl), TE buffer 18 μl, 10 times concentration of XbaI buffer were (NaCl 500 mM, tris · HCl (pH7.9) 200mM, MgCl 2 10
0 mM, 2-mercaptoethanol 100 mM) 2 μ
1, 1 μl of restriction enzyme XbaI (15 units) were sequentially added, and the reaction was carried out at 37 ° C. for 2 hours. This DNA solution was mixed with an equal volume of a mixture of phenol and chloroform (phenol: chloroform: isoamyl alcohol = 25: 25: 1,
3% sodium acetate solution (pH 5.2) 1/1 after extraction with 0.1% quinolinol saturated TE buffer solution)
The DNA was precipitated by adding 0 volume and 3 volumes of ethanol. After washing this DNA with 70% ethanol, TE8μ
l of exonuclease I
II Buffer (Tris HCl (pH 8.0) 500
mM, MgCl ₂ 50 mM, 2-mercaptoethanol 100 mM) 1 μl and exonuclease III
[Nippon Gene Co., Ltd.] 1 μl (50 units) was added, and the reaction was carried out at 30 ° C. for 1 minute, and then immediately H ₂ O 24 was added.
μl and 10 times concentrated nuclease S1 buffer (sodium acetate (pH 4.5) 330 mM, NaCl 50
0 mM, ZnSO ₄ 0.3 mM) ₄ μl and nuclease S1 [Boehringer Mannheim Yamanouchi Co., Ltd.] 2 μl
(20 units) was added, and the mixture was reacted at room temperature for 15 minutes. After the reaction, the DNA was purified by phenol / chloroform extraction and ethanol precipitation in the same manner as described above.
It was dissolved in 23 μl. To this solution was added 10-fold concentrated M buffer (NaCl 500 mM, Tris-HCl (pH
7.5) 3 mM of 100 mM, MgCl ₂ 100 mM, dithiothreitol 10 mM and dNTP mixed solution (dGT
3 μl of 0.5 mM each of P, dCTP, dATP, and dTTP) was added, and then DNA polymerase I [l
1 .mu.l of the charge fragment, Takara Shuzo Co., Ltd.
Add (4 units) and let react at 37 ° C for 30 minutes.
The NA end was blunted. Phenol / chloroform,
After purification by ethanol precipitation, the DNA obtained was
It was dissolved in μl of TE buffer to obtain a vector solution.
On the other hand, the recombinant plasmid p53A1 obtained in Example 4
A DNA fragment of 1.8 kb containing acyA, which was obtained by digesting 00 μg with restriction enzymes SalI and BamHI, was gene-cleaned [Gene clean (Bio10.
1)] and separated and purified to obtain about 4 μg of the target fragment. This DNA fragment was blunt-ended under the same conditions as above, and then purified by phenol / chloroform extraction and ethanol precipitation, and a TE solution (0.2 μg
/ Μl). Then 2.5 μl of this solution (0.5 μl
g), 2.5 μl (0.5 μg) of the above vector solution,
Purified 13 μl, 10 times concentration of ligation buffer (Tris.HCl 500 mM, pH 7.9), MgC
l ₂ 100 mM, dithiothreitol 200 mM, AT
P100 mM) 2 μl and T ₄ DNA ligase [Nippon Gene Co., Ltd.] 1 μl (300 units) were sequentially added,
The reaction was carried out at 16 ° C for 15 hours. Using 2 μl of this reaction solution, 100 μl of E. E. coli JM103 competent cells were transformed. This method is
niatis et al. (Molecular Cloning), Cold Spring Harbor, Laboratory (Cold Spri).
ng Harbor, published by Laboratory). The plasmid possessed by the transformant thus obtained was extracted and examined by agarose electrophoresis.
A plasmid pAB5 having an insert of about 1.8 kb having the structure shown in FIG. 7 was obtained.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0089

[Correction method] Change

[Correction content]

(B) 10 μg of pAB10 and pAB10ΔB1 plasmid pAB5 were completely digested with the restriction enzyme SacI, followed by phenol / chloroform extraction and ethanol precipitation to purify the DNA fragment. Next, of the fragments obtained by partial digestion with the restriction enzyme PstI, only the DNA fragment derived from pUC18 was excised, and the resulting DNA fragment of about 9.2 kb was separated by 0.8% agarose gel electrophoresis. Gene C
It was recovered and purified using lean (Bio101). This DNA fragment was dissolved in 22 μl of TE buffer, and
Double concentration of T ₄ polymerase buffer (Tris ・ HCl
(PH 7.4) 700 mM, MgCl ₂ 100 mM, dithiothreitol 50 mM 3 μl and dNTP mixed solution (d
0.5 mM each of GTP, dCTP, dATP, and dTTP
3 μl of the mixed solution) was added, and then 2 μl (4.8 units) of T ₄ DNA polymerase [Toyobo Co., Ltd.] was added and reacted at 37 ° C. for 30 minutes to blunt the DNA ends. After phenol / chloroform extraction and purification by ethanol precipitation, the product was dissolved in 45 μl of TE buffer and dissolved in 5 μl of 10-fold ligation buffer and T ₄ DNA ligase [Nippon Gene Co., Ltd.] 1 μl.
(300 units) was added, and the mixture was reacted at 16 ° C. for 15 hours for self-cyclization. After the reaction, 20 μl of the reaction solution was used to produce Streptomyces lividans (Strepto
Transformation of protoplasts of myces lividans TK2 4 was performed. A plasmid in which pUC18 portion was completely deleted was selected from the obtained transformants based on the difference in the electrophoretic pattern, and pAB10 having the structure shown in FIG. 18 and the structure shown in FIG. 19 were selected from these plasmids. pAB10ΔB1 was obtained. Both plasmids are pA
This is the same in that the pUC18 region is deleted from B5, but in the latter (pAB10ΔB1), a side reaction of the enzyme results in a deletion that extends to the 4 "-position acylase gene acyB region. Cannot produce acyl acylase.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0090

[Correction method] Change

[Correction content]

(C) Production of 3-O-Acetyltylosin One platinum loop amount of a slant culture of Streptomyces fradeier ATCC 19609 was added to GPY medium (1.0% glucose, 0.4% polypeptone, 0.4 yeast extract).
_{%, MgSO 4 · 7H 2 O} 0.05%. K ₂ HPO ₄
0.1% and glycine 0.05%, pH 7.2) 20
Inoculate a 250 ml Erlenmeyer flask containing ml,
The cells were shaken at 48 ° C. for 48 hours. 0.5 ml of this culture was inoculated into a 250 ml Erlenmeyer flask with a spring containing 25 ml of YEME + 15% sucrose medium (containing 5 mM MgCl ₂ and 0.5% glycine) shown in Example 3 and shaken at 28 ° C. for 40 hours. Cultured. The cells were collected by centrifugation (3,000 rpm, 15 minutes), washed with 10 ml of 10.3% sucrose, well suspended in 4 ml of P medium containing 1 mg / ml lysozyme, and reacted at 30 ° C for 1 hour. I went. The composition of P medium is as shown in Example 4. After the reaction, the remaining hyphae are removed by cotton filtration and centrifuged (3,000 rp
m, 10 minutes) to collect protoplasts 3 × 10 ⁹
The cells were suspended in P medium so that the number of cells / 100 μl. The above-mentioned pAB10ΔB1 solution (15 μg / 10 μl)
10 μl was added, and transformation was carried out in the presence of 25% PEG1000 [BDH Chemical (Chemical)]. The transformed protoplasts were washed with P medium and then resuspended in 1 ml of P medium. That 0.1m
1 to R3 medium (13.5 g of sucrose, KCl
0.05 g , glucose 1.0 g, polypeptone 0.4
g, yeast extract 0.4g, TES buffer 0.573
g, 10% MgCl ₂ solution 6.1 ml, Bacto agar 2.0 g and distilled water 100 ml; KH ₂ PO ₄ (0.5%) 1.0 ml, CaCl ₂ (5) after autoclave sterilization
M) (0.3 ml and 1.8 ml of NaOH (1N) was added), and the mixture was cultured at 28 ° C. for 16 hours. After culturing, the final concentration of thiopeptin is 5μg / ml or 25μ
The soft agar medium containing thiopeptin was overlaid so that the concentration became g / ml, and the cells were further cultured at 28 ° C. for 7 days. Soft agar medium is Nutrient broth
Oth) (8 g) and Bact agar (3 g) were added with distilled water (1 l), and autoclaved at 120 ° C. for 15 minutes.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0098

[Correction method] Change

[Correction content]

(E) Transformation with pAB10 One platinum loop of a slant culture of Streptomyces fradeier ATCC19609 was cultured in the same manner as in (C) , and protoplastized with lysozyme. As a plasmid, pIJ702 (from Streptomyces lividans ATCC35287, Hopp
the method of wood [Genetic Manipulation of Streptomyces (Genetic Ma
nipolation of Streptomyce
s): A Laboratory Manual (A Labora)
Tory Manual, John Yonnes Foundation (John Inns Foundation)
on, Norwich, UK, 198
5 year]), and the resulting transformant was protoplastized and regenerated by the same method as in the item (C) , and the strain (S. fraddi lacking pIJ702 was regenerated.
aeC-1) was obtained.

[Procedure correction 6]

[Document name to be amended] Statement

[Correction target item name] 0099

[Correction method] Change

[Correction content]

The S. fradiaeC-
1 was cultured and protoplasted in the same manner as in (C) ,
The above-mentioned plasmid pAB10 solution (15 μg / 10 μ
l) 10 μl was added, and transformation was carried out in the presence of 25% PEG1000 [BDH Chemical (Chemical)]. The transformed protoplasts were washed with P medium and then resuspended in 1 ml of P medium. The 0.
1 ml of R3 medium [Syucrose 13.5 g, KCl
0.05%, glucose 1.0 g, polypeptone 0.
4g, yeast extract 0.4g, TES buffer 0.57
3 g, 10% MgCl ₂ solution 6.1 ml, Bacto agar 2.0 g and distilled water 100 ml; KH ₂ PO ₄ (0.5%) 1.0 ml, CaCl after autoclave sterilization
0.3 ml of ₂ (5M) and 1.8 ml of NaOH (1N)
And then added] and cultured at 28 ° C. for 16 hours.
After culturing, the final concentration of thiopeptin was 5 μg / ml or 2
Soft agar medium containing thiopeptin was overlaid so that the concentration became 5 μg / ml, and the cells were further cultured at 28 ° C. for 7 days. Soft agar is Nutrient Broth
broth) 8g and Bact agar 3g 1l distilled water
Was added and autoclave sterilization was performed at 120 ° C. for 15 minutes.

[Procedure Amendment 7]

[Document name to be amended] Statement

[Correction target item name] 0103

[Correction method] Change

[Correction content]

(F) Confirmation of 3-O-acetyl-4 ″ -O-isovaleryl tylosin The above-mentioned production culture medium 1 l culture medium (50 ml medium 2)
(0 main culture), extraction operation was performed with ethyl acetate at pH 8.5. The solution was redissolved in 200 ml of acidic water (0.01N HCl), adjusted to pH 8.5 again, and extracted with 50 ml of ethyl acetate. This extract was concentrated to 10 ml, and spotted on each of 5 thin layer plates (Art 5715 manufactured by Merck [Merck]) in an amount of 2 ml in a strip shape and developed with the above-mentioned developing solvent. After air-drying, the silica gel in the spot portion of Rf = 0.82 was scraped off and extracted with 10 ml of acetone. The extract was evaporated to dryness in a vacuum evaporation concentrator to obtain 5 mg of white powder. The results of examining the physicochemical properties of this substance are shown below.

[Procedure amendment]

[Submission date] June 7, 1991

[Procedure Amendment 1]

[Document name to be corrected] Drawing

[Name of item to be corrected] Fig. 18

[Correction method] Change

[Correction content]

FIG. 18

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication C12N 9/18 (C12N 15/54 C12R 1: 465) (C12P 19/62 C12R 1: 465) ( (C12P 19/62 C12R 1:54) (C12N 1/21 C12R 1: 465) (72) Inventor Kazuhiko Okamura 2502-1 Fujisawa, Fujisawa-shi, Kanagawa (72) Inventor Hiroshi Tone 3-Namiki, Kanazawa-ku, Yokohama-shi, Kanagawa 7-4- 1003 (72) Inventor Rokuro Okamoto 2-18 Hananoki, Fujisawa City, Kanagawa Prefecture

Claims (30)

[Claims] 1. A macrolide antibiotic having the size of about 1.8 kilobases (kb) derived from a strain belonging to the genus Streptomyces and characterized by the restriction enzyme map shown in the following figure. DNA fragment containing gene acyA encoding the acylase of Escherichia coli and its restriction enzyme D
NA restriction fragment. Restriction map [Chemical formula 1] 2. A strain belonging to the genus Streptomyces is Streptomyces ambofaciens.
ambofaciens), Streptomyces kitasatoensis, Streptomyces narvonensis var josamyceticus, Streptomyces hygroscopicus, S. hygroscopicus S. platensis, S. albireticul
i), Streptomyces cinerochromogenes (S. ci
nerochromogenes), S. djakartensis (S. djakartensis), S. furdicidicus, S. macrosporeus, S. tendae ), Streptomyces thermotolerans,
And a DNA restriction fragment according to claim 1, which is selected from S. deltae and Streptomyces deltae. 3. A strain belonging to the genus Streptomyces is S. thermotoler.
ans) ATCC 11416 DNA
DNA restriction fragment by the fragment and its restriction enzyme. 4. The DNA fragment according to claim 1, wherein the macrolide antibiotic is selected from tylosin, spiramycin, leucomycin and nidamycin, and a DNA restriction fragment thereof with a restriction enzyme. 5. The DNA fragment according to claim 4, wherein the macrolide antibiotic is tylosin, and the DNA restriction fragment with a restriction enzyme thereof. 6. The following amino acid sequence: The DNA fragment according to claim 1, which comprises a DNA portion encoding the DNA fragment, and a DNA restriction fragment thereof with a restriction enzyme. 7. The following base sequence: [Chemical 4] [Chemical 5] A DNA fragment according to claim 1 having the following: and a DNA restriction fragment thereof with a restriction enzyme. 8. A macrolide antibiotic at position 3 having a size of about 1.8 kilobases (kb) derived from a strain belonging to the genus Streptomyces and characterized by the restriction enzyme map shown in the following figure. DNA fragment containing gene acyA encoding the acylase of Escherichia coli and its restriction enzyme D
NA restriction fragment. Restriction enzyme map [Chemical formula 6] 9. The DNA according to any one of claims 1 to 8.
A plasmid containing the fragment. 10. pIHY-17, pIJ922, pIJ943, pIJ350, p
The DNA according to any one of claims 1 to 8, which is a vector plasmid selected from IJ702, pHC79, pUC18 and pUC19.
The plasmid according to claim 9, which is a recombinant plasmid incorporating the fragment. 11. The plasmid according to claim 10, wherein the recombinant plasmid is p53A. 12. A microorganism transformed with the plasmid according to any one of claims 9 to 11. 13. The microorganism according to claim 12, wherein the host microorganism is Streptomyces lividans TK24. 14. The microorganism according to claim 12, wherein the host microorganism is a macrolide antibiotic-producing bacterium that does not substantially produce the acylating enzyme at position 3 of the macrolide antibiotic. 15. The macrolide antibiotic-producing bacterium is Streptomyces fradiae A.
The microorganism according to claim 14, which is TCC 19609. 16. The method according to claim 12, wherein the microorganism according to any one of claims 12 to 15 is cultured in a medium containing a macrolide antibiotic, and the produced macrolide antibiotic acylated at the 3-position is collected. Method for producing 3-acylated macrolide antibiotic. 17. A method for producing a 3-acylated macrolide antibiotic, which comprises culturing the microorganism according to claim 14 in a medium and collecting the resulting macrolide antibiotic acylated at the 3-position. 18. A method for producing 3-O-acetyltylosin, which comprises culturing the microorganism according to claim 15 in a medium and collecting the produced 3-O-acetyltylosin. 19. A macrolide antibiotic is reacted with an acylating agent in the presence of the microbial cell according to any one of claims 12 to 15 or a treated product thereof.
-Method for producing an acylated macrolide antibiotic. 20. A gene DNA having the ability to acylate the DNA fragment of claim 1 or 8 and a macrolide at the 4 "-position.
A recombinant plasmid in which a fragment and a vector plasmid are incorporated. 21. The vector plasmid is pIHY-1.
7, pIJ922, pIJ943, pIJ350, pI
The recombinant plasmid according to claim 20, which is selected from J702, pHC79, pUC18 and pUC19. 22. The plasmid according to claim 20 or 21, wherein the recombinant plasmid is pAB10. 23. A microorganism transformed with the plasmid according to any one of claims 20 to 22. 24. The microorganism according to claim 23, wherein the host microorganism is Streptomyces lividans TK24. 25. The macrolide antibiotic-producing bacterium, wherein the host microorganism does not substantially produce the acylating enzymes at the 3rd and / or 4 ″ positions of the macrolide antibiotic.
Microorganisms described. 26. The macrolide antibiotic-producing bacterium is Streptomyces fradiae AT
The microorganism according to claim 25, which is CC19609. 27. Culturing the microorganism according to any one of claims 23 to 26 in a medium containing a macrolide antibiotic, and collecting the produced macrolide antibiotics having acylated positions 3 and 4 ". 3- and 4 "-characterized by
A method for producing an acylated macrolide antibiotic. 28. The microorganism according to claim 25 or 26 is cultivated in a medium, and the produced macrolide antibiotic having acylated positions 3 and 4 ″ is collected. A method for producing an acylated macrolide antibiotic. 29. The microorganism of claim 26 is cultured in a medium, and the produced 3-O-acetyl-4 ″ -O-isovaleryl tylosin is collected. A method for producing -O-isovaleryl tylosin. 30. A macrolide antibiotic is reacted with an acylating agent in the presence of the microbial cell according to any one of claims 23 to 26 or a treated product thereof.
-And 4 "-Methods for producing acylated macrolide antibiotics.