JPS62224292A - Recombinant dna containing cephamycin c producing gene, transformant containing same and production thereof - Google Patents

Abstract

PURPOSE:To obtain cephamycin C in large production, by cultivating a bacterium belonging to the genus Streptomyces, containing specific genetric recombinant DNA in a medium. CONSTITUTION:Chromosome DNA of a bacterium such as Streptomyces cattleya, etc., capable of producing cephamycin C is cleft with restriction enzyme Sau3A to give DNA fragment of 20-40Kb. Then, this fragment is inserted into a cleavage site of plasmid vector to give recombinant DNA, which is blended with a protoplast such as Streptomyces lividans, etc., as a host in the presence of polyethylene glycol to give a bacterium capable of producing cephamycin C, a transformant. Then, the bacterium is cultivated in a medium containing glucose, peptone, salt, etc., at pH of weak acidity - neutrality at 23-30 deg.C by aerated stirring to give a culture mixture, which is treated by solvent extraction, affinity chromatography, etc., to collect cephamycin C.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a recombinant DNA containing a cephamycin C-producing gene obtained by genetic engineering, and a transformant transformed with the recombinant DNA. The present invention relates to a transformant and a method for producing cephamycin C using the transformant.

[Conventional technology]

There are currently over 5,000111 antibiotics known, most of which are produced by actinomycetes. Among these antibiotics, semi-synthetic β-lactam antibiotics are particularly widely used due to their toxicity, efficacy and suitability for chemotherapy. These useful antibiotics are secondary metabolites that are not directly essential to the producing countries, and their production amounts are extremely small. Therefore, in order to increase the production amount, great efforts have been made to improve microorganisms by improving the culture medium and culture conditions, and by inducing mutagenesis.

On the other hand, in recent years, genetic engineering techniques have been established to produce large quantities of useful trace amounts of physiologically active proteins using microorganisms such as Escherichia coli. For this reason, genetic engineering techniques are used in various fields, and this technology is also being used in the production of antibiotics.

For the application of genetic engineering techniques in actinomycetes, see Bibb et al.
ature.

274, 398-400.1978), and recently Hompwood, Omura et al.
opwood.

et al., Nature, 314.642-
644, 1985) has been reported to produce a novel antibiotic medelrosin A. Also, Baizeau et al. (Ba1
ley+ C,R,,et allBiotechno
logy, 808-811 + Septembe
r + 1984) also reported on the cloning of the clavulanic acid producing gene.

[Problems to be solved by the present invention]

However, since antibiotics are secondary metabolites, many genes are involved in their production, and there are still a limited number of antibiotics that can be used with genetic engineering techniques. Also,
The only known gene for producing β-lactam antibiotics, which are the main chemotherapeutic agents, is the cloning of the claplanic acid gene by Beizo et al.

[Means for solving problems]

As a result of intensive research into the production of β-lactam antibiotics by actinomycetes using genetic engineering methods, the present inventors succeeded in obtaining a new cephamycin C-producing bacterium and completed the present invention. reached.

That is, the present invention provides a recombinant DNA containing a cephamycin C-producing gene obtained by genetic engineering, a transformant transformed with the recombinant DNA, and a cephamycin C-producing gene using the transformant. Relating to a manufacturing method.

The present invention will be explained below.

(1) Method for preparing a cephamycin C-producing gene from chromosomal DNA The cephamycin C-producing gene used in the present invention can be obtained from a microorganism capable of producing cephamycin C. For example, actinomycetes belonging to the genus Streptomyces, specifically Streptomyces clavuligerus (
Streptomyces clavuli-geru
s), Streptomyces lactamdulans (
S. lactamdurans), Streptomyces lactamgens (S. lactamgens)
, Streptomyces wadayamensis (S, wa
dayensis), Streptomyces albogriseolus (S.

albogriseolus), Streptomyces inositovorus (S, 1nosttovorus)
), Streptomyces jumondinensis (S
, jumonj 1nensis), Streptomyces todrominensis (S, todo-romine
nsis), Streptomyces philippinensis (S, Fi. pinensis), Streptomyces cattleya (S, cattleya), and the like.

As a method for obtaining the cephamycin C-producing gene from these bacterial cells, first, a known method (Chater et al.

K, F, et al, Current
Topics in Microbiol.

and Immunol, 96, 69-95.
6. Next, the prepared chromosomal DNA is cut into fragments by restriction enzymes. The restriction enzyme that can be used for this cleavage is preferably one that i) can cleave chromosomal DNA, and ii) does not cleave the cephamycin C-producing gene midway through. Furthermore, even with a restriction enzyme that cuts the cephamycin C-producing gene in the middle, a preferable DNA fragment can be obtained by partially digesting the gene by changing the reaction conditions.

Genes involved in the production of antibiotics are classified into gene groups ((Gen
It is known that e clusters are formed (Malpavtjda, F., et al.
, Nature, 309+462-464.198
4. (or see the above-mentioned paper by Beizeau et al.) Therefore, the DNA fragment needs to have a relatively large molecular weight.

The obtained DNA fragments are separated by known methods (eg, agarose gel electrophoresis and sucrose density gradient method).

In the present invention, a DAN fragment consisting of 20-40 kb base pairs was isolated by cutting chromosomal DNA obtained from Streptomyces Cattleya with the restriction enzyme Sau 3A.

(2) Selection of host-vector group Examples of vectors include plasmid vectors and phage vectors, preferably those into which a DNA fragment can be inserted, and which can be retained or expressed in the host or transformant. . More preferred are those that can replicate within the genus Streptomyces and have selection markers such as drug resistance, pock formation, and auxotrophy to detect transformants. . Plasmid vectors include 5CP2'', 5LP1.2,
pIJlol, plJ102, pIJ41,
Examples of phage vectors include plJ61, plJ702, plJ942 and derivatives thereof, and phage vectors include φC31 and derivatives thereof. Since the vector is usually conserved in the host microorganism, for example the method of Charter et al. or the method of Kaiser et al. (Plasmid,
12.19-36.1984).

The isolated vector can be cleaved using an appropriate restriction enzyme.

As a host, 1) it must be able to be made into a protoplast for transformation etc. and has good regeneration efficiency; ii) it must have a weak or no restriction modification system; and iii) it must be well maintained when introducing a vector. It is preferable that the following conditions are satisfied.

If these conditions are not met, the strain must be improved, which can occur naturally or by mutagenesis. Furthermore, in the present invention, either a host that does not produce the target antibiotic or a host that does produce the target antibiotic can be used.

In the present invention, the thiostrepton resistance gene (tsr) and melanin pigment production gene (m
It is preferable to use plJ943, which has the selection marker el) and a restriction enzyme BgI n cleavage site for inserting a DNA fragment into the melanin pigment producing gene.

In addition, as a host, Streptomyces lividans (S, liν1-da
ns) wo, Streptomyces lactamgens (S, 1actamgens) is a cephamycin C-producing bacterium.
) U-108 (Ferm-P2251) is preferably used.

(31) Preparation of Jl recombinant DNA The recombinant DNA is prepared by the usual method in this field (
Maniatis, T., et al. + Mol
ecularcoloning, a Laborat
ory manual+ Co1d SpringHo
rbor Laboratory, New York
and the above-mentioned literature by Baytwo et al.). More specifically, (
The DNA fragments obtained in 1) are combined and integrated in the presence of glue such as T4 ligase. On this occasion,(
Appropriate reaction conditions are selected depending on the type of vector and restriction enzyme used in methods 1) and (2).

(4) Transformation method The recombinant DNA obtained by the above method is introduced into a host, and the transformation of actinomycetes is performed using protoplasts. Protoplasts are preferably obtained by culturing bacterial cells in a normal medium and treating them with lysozyme at an appropriate time for protoplast formation. At this time, by adding glycine to the medium, bacterial cells with high lysozyme transferability can be obtained (Okanishi, et al.
al., J. Gen.

Microbiol, +80, 389-400.1
974 and Charter et al., cited above). In order to form protoplasts, it is necessary to melt the cell wall under mild conditions and to stably maintain the formed protoplast in a state where it can reproduce. For protoplast stabilization, isotonic agents (such as sig sugars) and divalent metal ions (such as calcium or magnesium ions) are recommended.
is necessary.

Transformation was performed in the aforementioned Bibb et al. paper and in Thompson et al.
Bacteriol,, 151.668-677,
(1982). Specifically, this is carried out by mixing protoplasts and Mi recombinant DNA in the presence of polyethylene glycol. When using a phage vector, the efficiency of transformation may be improved by embedding the m-recombinant DNA in liposomes.

The transformants thus obtained are isolated and selected by primary selection using a marker possessed by the vector and by using the production of the desired antibiotic as an indicator.

In the present invention, as a secondary selection, thiostrepton-resistant and non-melanin producing cells are isolated, and as a secondary selection, β
- Co+mamonas Terrigena sensitive to lactam antibiotics
) Bioautography using M2 (Bioout
Transformants P
F15-1 was obtained. This transformant is a national
Collection of Industrial and Marine Bacteria (National Col.
ion of Industrial and Mar
ine Bacteria: NCIB), 19
It was deposited on March 19, 1986 under deposit number NCI 812227. In addition, a recombinant DNA containing the cephamycin C-producing gene was isolated from the transformant PF15-1 and named pPF900. , Mi recombinant DNA
pPF900 consists of 49.9 kb base pairs and has a restriction enzyme cleavage map as shown in FIG. In the figure, plJ900 is DNA derived from plJ943 (2
0.6 kb: indicated by a thin line in the figure) and Streptomyces cadrea NRR involved in cephamycin C production
DNA derived from L8057 (29.3 kb: in the figure,
(shown with a thick line).

(5) Production of produced antibiotic The transformant thus obtained is cultured according to the culture method for general actinomycetes, preferably by shaking or aerated agitation culture in a liquid medium. As the medium components, those known as nutritional sources for actinomycetes are used, such as glucose, glycerin, maltose, and carbon sources.
Sig sugar, dextrin, starch, etc. are nitrogen sources, soybean flour, cottonseed flour, peptone, meat extract, corn staple liquor, etc., and inorganic salts are table salt, phosphoric acid,
Calcium carbonate, etc., and trace metals such as ferrous sulfate,
Magnesium sulfate, zinc sulfate, manganese chloride, cobalt chloride, etc. are added as appropriate. The pH of the medium is preferably slightly acidic to neutral, and the culture temperature is preferably 23 to 30°C.

The antibiotic produced in the culture medium can be recovered according to a general antibiotic collection method.

i.e., the culture was subjected to solvent extraction, precipitation and ion exchange,
Antibiotics can be recovered by treatment with various chromatography methods such as partitioning, gel filtration, and affinity.

Confirmation of antibiotics in the culture medium or during the extraction and purification process can be performed by bioautography using a paper disk method or bioautography using thin layer chromatography (TLC). In addition, in purified samples, the produced antibiotic can be identified by known methods such as melting point, ultraviolet absorption spectrum, infrared absorption spectrum, X''&'31&'31 diffraction chromatography, and high performance liquid chromatography (HPLC). Ru.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to these in any way.

Example 1 Preparation of Niblasmid Vector (plJ943) Streptomyces lividans (S, l1vidans) 1326 containing plasmid plJ943
Tryptone Soya Broth (Tryptone 5oya B) supplemented with thiostrepton 10μg/ml
roth: manufactured by Difco) at 30°C in 50ml
Cultured for 24 hours. 14 bacterial cells (approximately 2 g as wet weight)
Bacteria were harvested by centrifugation at 0xg, followed by
．． Washed with 3M sucrose. 0.5g of bacterial cells was mixed with 0.3M sucrose and 25mM ethylene dinitrilotetraacetate (
EDTA), RNase A50, Ug/ml
and lysozyme 2 rn
g/m! 25mM Tris-HCl buffer (pH 8) containing 2
．． ' 5 ml.

After incubation for 1 hour at 37°C, the bacterium was lysed by mixing with 1.25 ml of 0.3 M sodium hydroxide containing 2% sodium dodecyl sulfate (SDS), and
Incubated for 30 minutes at °C. Add 0.4 ml of a mixture of phenol and chloroform (1:1) to the lysate, stir the mixture well at room temperature, and then add 4500 x g
Centrifuged for 15 minutes.

The upper aqueous layer is collected and the extraction is repeated with the mixture of phenol and chloroform until proteinaceous aggregates in the middle layer are no longer visible. Finally, an equal amount of isopropanol was mixed with the collected aqueous layer and left at -20°C for 1 hour. The precipitate containing plasmid DNA was washed at 15,000×g for 1
Collected by centrifugation for 0 minutes, and diluted with 10
The mixture was redissolved in 2.5 ml of Lis-HCl buffer (pH 8; hereinafter abbreviated as TE buffer). Approximately 4 μg of plasmid plJ943 was obtained by this method.

Example 2: Preparation of chromosomal DNA Streptomyces cadrea (S, cat, tle
ya) NRRL8057 with 0.4% peptone, 0.4%
Yeast extract, 0.4% magnesium sulfate heptahydrate, 0
．． 50 ml of liquid medium containing 0.05% potassium dihydrogen phosphate, 0.2% potassium hydrogen phosphate and 1% glucose
The cells were cultured for 24 hours at 27°C. Bacterial cells were collected by centrifugation at 1400×g and washed with 0.3M sucrose. 0.5g of bacterial cells
0.3M glucose, 25mM EDTA, RN a
s e A 50 ug/ml and lysozyme 2
25mM Tris-HCl buffer containing @II/ll11 25
It was suspended in ml. After incubating at 37°C for 1 hour, the cells were lysed by mixing with 1.25 ml of 2% SDS.
Thereafter, it was incubated at 37°C for 30 minutes. The lysate was gently mixed with 3.75 ml of phenol previously saturated with 0.1 M Tris-HCl buffer (pH 8) at room temperature for about 10 minutes, and then centrifuged at 4500xg for 15 minutes. The upper aqueous layer is collected and the extraction is repeated with phenol saturated with 0.1 M) Lis-HCl buffer (pH 8) until proteinaceous aggregates in the middle layer are no longer visible.

The finally collected aqueous layer was mixed with an equal amount of isopropanol and left at -20°C for 1 hour. The precipitate containing chromosomal DNA was collected by centrifugation at 15,000×g for 10 minutes, and TE
It was redissolved in 2.5 ml of buffer.

This method yields approximately 150 I! of chromosomal DNA! g was obtained.

Example 3 Restriction enzyme BglI of Niblasmid plJ943
9431 μl of the plasmid p I J obtained according to Example 1 was added to 50 ttl of Lis-HCl buffer (pH 7,4) containing 100 mM sodium chloride, 10 mM magnesium chloride, lQmM2-mercaptoethanol and 100 μg/ml bovine serum albumin. Then, 2 units of restriction enzyme BgllI were added and incubated at 37°C.
Incubated for 2 hours.

Example 4: Cleavage of Chromosomal DNA 1 containing 100mM sodium chloride, 10mM magnesium chloride and 100 pg/ml bovine serum albumin
0mM) Liss-HCl buffer? pi, (pH 7,5) 100μ
1, Streptomyces obtained in Example 2
3 μg of Cadrea chromosomal DNA was added, followed by 5 au3A1/8 units of restriction enzyme, and the mixture was incubated at 37° C. for 1 hour. Thereafter, 100 μl of phenol previously saturated with 0.1 M ToIJ salt MrA loading solution (pH 8) was added, mixed well for 10 minutes at room temperature, and centrifuged at 4500×g for 15 minutes. Collect the upper aqueous layer, mix with 250 μl of isopropanol and incubate at -20°C for 1 hour.
I left it for a while. The DNA precipitate was collected by centrifugation at 15,000xg for 10 minutes, redissolved in 0.3 ml of TE buffer,
40% containing 5mM sodium acetate, 1mM EDTA
0.6% in mM Tris-acetate running buffer (pH 8)
Electrophoresis was performed on agarose.

The agarose gel portion containing the DNA fragment having base pairs of 20-40 kb was cut out and placed in a dialysis bank containing approximately 1 ml of running buffer. DNA fragment is 5pol) / c
The mixture was electrophoresed for 1 hour at 100 ml of phenol, extracted with 1 ml of phenol, and precipitated with isopropanol. This precipitate is T
Redissolved with 10μ2 of E buffer.

Example 5: Binding of chromosomal DNA fragment to plasmid (preparation of recombinant DNA) 5 μg of chromosomal DNA fragment obtained in Example 4 and plasmid plJ943B obtained in Example 3
0.5 μg of gllll fragment was mixed with 10 mM magnesium chloride, 10 mM dithiothreitol and 0.6 mM
20mM containing adenosine triphosphate (ATP)
) Binding is carried out by incubation with 0.5 units of T4 ligase in 300 μβ of Lys-HCl buffer (pH 7,6).

After incubation at 16°C for 3 days, the DNA was precipitated with isopropanol and the precipitate was diluted with 0.6 M sucrose 1
It was redissolved in a mixture of 0 μl and 10 μl of TE buffer.

Example 6: Host protoplastization of Streptomyces lividans (S, l1vi-d
ans) 1326 with 0.3% yeast extract (Difc
(manufactured by Dffco), 0.5% peptone (manufactured by Dffco), 0.5% peptone (manufactured by Dffco), 0.5% peptone (manufactured by Dffco),
3% malto extract (manufactured by Difco), 1% glucose, 34% Siq $Jj, 0.5% glycine and 5mM
30'C1 in medium 50111 containing magnesium chloride
Cultured for 64 hours. Bacteria were collected from 5 ml of the culture medium by centrifugation at 1400xg and washed with P buffer. The composition of the P buffer is shown below.

Composition of P buffer aP, 103 g)
hsO40,25g MgC1z ・61120 2
．． 03 gl trace element solution 2
ml (Trace Element 5olutio
n) Tea water 900 ml After sterilizing the above composition in an autoclave at 121°C for 20 minutes, the following substances are added.

5% KHtPOa 1 ml
7.36% CaC1g ・2Hz0
50 mlO, 5M N-1-lis(hydroxy) 50 ml methyl-2-aminoethanesulfonic acid 0 Trace Elem solution
entSolution) composition ZnCIz 40
wFeCl: + '6HzO200ytCuClz
・2Hz0 10 KMnC
lz ・4Hz0 101W
NaJ40t ・1011z0
10 If(Nl14)aMOtOzn' 411
zO10mg distilled water 100
0 ml of washed bacterial cells was added to P-loose containing 1+ng/ml of lysozyme.

The cells were resuspended in 5 ml of liquid and incubated at 30°C.

After 1 hour, undigested bacterial cells and produced protoplasts were precipitated, washed twice with P buffer, and resuspended in 4 ml of P buffer. This suspension was centrifuged at 600×g for 10 seconds to precipitate bacterial cell fragments.

3.3 m of supernatant with approximately 5 x 10” protoplasts
This time, the cells were centrifuged at 600×g for 7 minutes to precipitate the protoplasts.

Example 7: Preparation and selection of transformants The protoplasts obtained in Example 6 were added to 3 m of 'IJ buffer solution containing 25% polyethylene glycol 1000.
l, 0.6M! 1 sugar and 10 μβ of the recombinant DNA prepared according to Example 5.

The composition of this Tilt solution is shown below.

Composition of T buffer 1M Tris salt # (pH 7,9) 50 m
lshi! Its! 25
gCaClz ・2Hz0 1
4.7 gKtSOa
O, 25g trace element? Liquid 2
ml distilled water 948
ml of the above composition is sterilized in an autoclave at 121°C for 20 minutes.

One minute later, the mixed solution containing the transformed protoplasts is diluted with P buffer, inoculated onto a regeneration medium (hereinafter abbreviated as R medium), and cultured at 30° C. overnight. The composition of this R medium is shown below.

Composition of R medium KzSO40, 25g MgCh ・611□0 10.
12 g glucose 10
g bacto-casamino acid 0.1' g
(manufactured by Difco) Bact yeast extract 5g (manufactured by Difco)
103 g agar
20g distilled water
After sterilizing 940 ml of the above composition in an autoclave at 121° C. for 20 minutes, the following sterilized solution was added.

Trace element solution 2ml 15%
1lzP042 ml 29.5% CaC1z ・211!0 1
0 m120% L-proline 15
mlO, 7M TES buffer (pH 7,2) 3
5 m1O317% 2N hydroxylation of tyrosine 2.
4 ml sodium volume? At night, pour approximately 20 ml of the above R medium into a 9 cm petri dish and allow it to solidify. Before use, air dry the medium with sterile air to remove 15% moisture.

After incubation, soak each plate with 1 ml of 250 μA/ml thiostrepton. The cells were incubated at 30° C. for 3 to 4 days. In order to screen for strains producing β-lactam antibiotic 1llI, first, transformants that did not produce melanin pigment and showed resistance to thiostrepton were selected.

Example 8: Screening for β-lactam antibiotic-producing transformants The non-melanin pigment-producing, thiostrepton-resistant transformants selected in Example 7 were treated with 2% glycerin and 2.8% corn staple liquor. , 1% Tsurulac (5olu
lac: manufactured by Grain Processing), 1
．． 05% 3-morpholinopropanesulfonic acid ((MOP
SSpH7), 0.8% Proflo (Profl.

: Traders), cobalt chloride hexahydrate o,
ippm, 0.04% tyrosine, thiostrepton 2
It was inoculated onto a culture medium containing 0 μg (7 ml) and 2% agar, and cultured at 30° C. for 6 to 7 days, and the grown colonies were taken out as agar pieces. - Night-cultured Comamonas terrigena M
2 of 1150f and 2,3.5-triphenyltetrazolium chloride (TCC) IGIP
Antibiotic Medium I:
Difco company! ! j) Place the agar piece on top and heat at 30℃.
Cultured overnight. β-lactam antibiotic producing strains were screened by visually measuring the growth inhibiting activity of agar pieces containing the transformants against Comamonas terrigena M2.

As a result, a transformant that formed a growth inhibition circle against Comamonas terrigena M2 was confirmed, and this transformant was transferred to PF.
It was named 15-1. It was confirmed through various experiments in Example 11 that this PF15-1 produces cephamycin C.

Example 9: Analysis of recombinant DNA pPF900 Recombinant DNA was isolated from transformant PF15-1 in the same manner as in Example 1, and its effects on restriction enzymes, base pairing, etc. were examined. The isolated recombinant DNA showed a restriction enzyme cleavage map as shown in FIG.
29 consisting of k b base pairs and derived from the chromosomal DNA of Streptomyces cadrea NRRL8057.
．． pl into which a DNA fragment consisting of 3 kb base pairs was inserted.
It was analyzed that it was J943. This recombinant DNA
was named pPF900.

Recombinant DNA pPF900 was reintroduced into Streptomyces lividans 1326 by the same method as in Example 6. This new transformant is transformant P
It produced the same level of cephamycin C as F15-1.

From this, it was confirmed that recombinant DNA pPF900 has a DNA fragment responsible for cephamycin C biosynthesis.

Example 10: Transformant PF15-1 and recombinant DNA
Culture of pPF900 reintroduced transformant (11) One strain of the transformant reintroduced with the transformant PF15-1 obtained in Example 8 or the recombinant DNA pPF900 obtained in Example 9 was cultured with thiostrepton 20. l
The cells were cultured in R medium (see Example 6) supplemented with ag/a11. Spores from the slant were treated with 3% sig sugar, 1.5
% Turlac, 0.5% yeast extract powder, 0.5% corn gluten meal and thiostrepton 10μg
/ml in a 250 ml flask containing 25 ml of seed medium (adjusted to pH 7.0 before sterilization) and cultured with shaking at 27°C for 2 days. Add 1 ml of this culture solution to 2% glycerin, 2.8% corn staple liquor, 1% Tsurlac,! , 05% MOPS buffer, 0.8
%Proflo, cobalt chloride monohydrate 0.1 p p
Culture medium containing m and thiostrepton lOμg/ml
The cells were transplanted into a 250+++1 volume flask containing 20n+1 cells, and cultured with shaking at 27°C for 5 days. After completion of the culture, the culture medium was examined by bioautography using FjJJi chromatography (TLC) or high performance liquid chromatography (HPLC) to measure cephamycin C production ability.

Each test method is explained in Example 11 below.

The amount of cephamycin C produced by the transformant PF15-1 was 2.
4μg/la1, recombinant DNA pPF90
In the 0 reintroduction transformant, it was 20 IJg/ml.

(2) Transformant PF15-1 or recombinant DN
A. Example 1 One strain of pPF900 re-introduced prize transformant
The cells were cultured in the same seed medium as used in 0(1).

Add 1 ml of this +IHIWt to 2% glycerin and 2% Pharmamedia (Pharmasedia: Tra
ders), 0.2% yeast extract, 1.05% MO
The cells were transferred into a 250 ml flask containing 20 ml of culture medium (adjusted to pH 7, 0 before surface reduction) containing PSIJj buffer solution, 5 ppm of cobalt chloride liquid-liquid hydrate, and 100 ttg/ml of thiostrepton, and incubated at 27°C. It was cultured with shaking for 5 days. As a result, the amount of cephamycin C produced by the transformant PF15-1 in this medium was 17 μg/
m I, and 10 μg/ml in the recombinant DNA pPF900 reintroduced transformant.

(3) Streptomyces lactamgens (S, lactamgens) U-108 (Fe
rm-P2251). As a result, two transformants that were resistant to thiostrepton were obtained. These two thiostrepton-resistant transformants and the parent strain Streptomyces lactamgens U-108 were cultured in the medium used in Example 10 (21).As a result, in this medium, the parent strain Streptomyces lactamgens The amount of cephamycin C produced by U-108 is 102 μg.
On the other hand, recombinant DNA pPF9
In the two transformants that were re-introduced with 00, each strain was 25
It showed production of 7 μg/l and 358 μg/+al.

Example 11: Examination of antibody substance produced by transformant PF15-1 Transformant PF15-1 or Mi recombinant DNApPF
900 reintroduction Streptomyces lividans 1326
The β-lactam antibiotic produced in the culture solution of the transformant was investigated by the following biological and chemical tests.

a. Antibacterial spectrum When the antibacterial spectrum of various test bacteria was measured by the paper disk method, the results shown in Table 1 were obtained.

- In the table below, "++" means strong growth-inhibiting activity, "+" means growth-inhibiting activity, and "-" means no growth-inhibiting activity.

As a result, the culture solution of the transformant PF15-1 showed the same antibacterial spectrum as the cephamycin C preparation against various test bacteria. Similar results were also obtained with the culture solution of the transformant reintroduced with the recombinant DNA pPF900.

b, chemically or physically stable transformant PF15-1 or m recombinant DNApPF9
Antibiotics produced from 00 reintroduction transformants were examined for stability against hydroxylamine, cysteine, acid, alkali, penicillinase, or heat. Hydroxylamine treatment: 0.05 g of hydroxylamine
was dissolved in 10 ml of distilled water and purified with sodium hydroxide.
10μ of solution prepared in H6.5! The sample is 90μ! and incubated for 30 minutes at 27°C. For cysteine treatment, add 10 μl of 2% DL-cysteine to 90 μl of the sample.
and incubated for 30 minutes at 27°C. For acid treatment, the sample was adjusted to pH 2 with phosphoric acid and incubated at room temperature for 30 minutes. For alkaline treatment, the sample was adjusted to pH 12 with sodium hydroxide and incubated at room temperature for 30 minutes. Benicillinase treatment was carried out using 10μ/ml Bacillus cereus.
Add 10 μl of derived penicillinase type to 90 μl of sample and treat at 30°C for 30 minutes, or add 10 μl of penicillinase type
Enterobacter -derived penicillinase type [10 μβ] was added to 90 μ2 of the sample and incubated at 25° C. for 30 minutes. Further, heat treatment was performed by incubating μl of the sample at 85° C. for 1 hour.

After the above treatment, the anti-concavity spectrum of the sample was determined as in Example 11.
Measured according to method a.

As a result, the antibiotics produced from both were relatively stable against hydroxylamine, cysteine, acid, and penicillinase type (■), but were rapidly inactivated by heat, alkali, and penicillinase type (■). Stability experiments were also conducted on a mixture of cephamycin C standard and R medium, and the results were similar to the above.

c. Bioautography by TLC The culture solution of each transformant and cephamycin C preparation were prepared separately or mixed and placed on a silica gel TLC sheet (Merck DC-Aluf
i) acetonitrile:water (7:3), i)
i) Developed with two solvents: isopropanol:water (7:3). The unfolded sheet is Comamonas terrigena M.
The cells were covered with antibiotic medium (2) in which 2 had been implanted, and cultured overnight at 30°C, and Rf4rL was measured. As a result, the Rf values of the cephamycin C specimen and the culture solution of each transformant completely failed.

d6 high performance liquid chromatography (HPLC) The culture solution of each transformant and cephamycin C preparation were transferred to a reverse phase column (N
ucleosil 10μ-C18), 0.05M
It was measured by HPLC using potassium phosphate buffer < p ii 2.5).

The antibiotic produced by each transformant was not produced by the parent strain, Streptomyces lividans 1326, but behaved similarly to the cephamycin C preparation.

In addition, the HPLC peak of the transformant obtained by introducing the recombinant DNA and A into Streptomyces lactamgens U-108 increased to about three times that of the parent strain. Furthermore, these peaks exhibited different behavior from cephalosporin C and deacetylcephalosporin C.

[Brief explanation of drawings]

Figure 1 shows the restriction enzyme cleavage map of recombinant DNA pPF900, and Figure 2 shows the plasmid vector prJ.
943 is shown.

Claims (3)

[Claims] (1) Recombinant DNA into which a DNA fragment containing a cephamycin C-producing gene has been inserted. (2) A cephamycin C-producing microorganism belonging to the genus Streptomyces containing a recombinant DNA into which a DNA fragment containing a cephamycin C-producing gene has been inserted. (3) Cultivating a cephamycin C-producing microorganism belonging to the genus Streptomyces containing a recombinant DNA into which a DNA fragment containing a cephamycin C-producing gene has been inserted, and collecting cephamycin C from the culture. A method for producing cephamycin C.