JPH07265080A - Fosfomycin biosynthesis-related gene and new method for producing fosfomycin with the same - Google Patents

Abstract

PURPOSE:To provide a fosfonycin biosynthesis-related gene for obtaining fosfomycin from 2-hydroxypropyl phosphoric acid, the fosfomycin being an antibiotic useful in the field of medical treatments. CONSTITUTION:The DNA contains the gene of an epoxidized enzyme which is an enzyme in the fourth stage of the biosynthesis of fosfomycin existing in an approximately 23kb gene cluster cloned from the DNA of a fosfomycin- producing bacterium and codes for an amino acid sequence of the formula. The gene can be obtained by a sub-cloning means using as a DNA donor the Streptomycin wedmorensis NP71 pFBG 23 strain (FERM P-13838) which is a strain transformed with plasmid pFBG 23 into that the gene cluster is inserted.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to genes for two enzymes, methylating enzyme and epoxidizing enzyme, which are involved in methylation reaction and epoxidation reaction, which are important reactions in the biosynthetic pathway of the antibiotic fosfomycin. Is. The present invention also provides a novel method for producing fosfomycin using a recombinant microorganism transformed with a plasmid containing an epoxidase gene.

Methylating enzymes and epoxidizing enzymes are enzymes involved in the formation of a methyl group and an epoxy ring, which are structural features of fosfomycin. In particular, the epoxy ring structure is considered to be a particularly important structure in view of the antibacterial activity of fosfomycin since the antibacterial activity disappears when the ring is opened by hydrolysis. Therefore, the activities of methylating enzyme and epoxidizing enzyme are considered to be important factors in the process of biosynthesis of fosfomycin.

Since fosfomycin is a low molecular weight compound and has few steps of biosynthesis, fosfomycin using a gene cluster containing the methylation enzyme gene and the epoxidation enzyme gene and these fosfomycin biosynthesis related genes provided by the present invention. Elucidating the mechanism of expression and regulation of biosynthesis-related enzyme activity can be used not only for fosfomycin biosynthesis research, but also for productivity improvement research and manufacturing method improvement research of fosfomycin, which is an antibiotic useful in the medical field. is there.

Fosfomycin is a substance found as a product of Streptomyces genus actinomycetes, but as an industrial production method, a method of chemically epoxidizing cis-propenylphosphonic acid is currently used. In the chemical synthesis method, racemization occurs and optical resolution is required, so that the production yield is limited. Therefore, a method for producing fosfomycin using cis-propenylphosphonic acid as a substrate focusing on the structure specificity and selectivity of the biosynthetic reaction of a microorganism has been studied, and a method using a microorganism of the genus Penicillium or Servibrio has been reported. .

[0005]

BACKGROUND OF THE INVENTION Fosfomycin is an antibiotic found in a culture solution of Streptomyces fradiae and Streptomyces wedmorensis, and has another unique mechanism of action. It is widely used for various infectious diseases due to its lack of cross resistance with.

Further, since fosfomycin has a CP bond in which carbon and phosphorus are directly bonded, which is extremely rare as a natural product, how fosfomycin is produced is as follows.
So-called biosynthetic studies have been carried out in detail by the present inventors, and it has been reported that they are produced from phosphoenolpyruvate in a four-step reaction. (S.Imai et al .: Agric.
Biol. Chem., 49, 873 (1985), H. Seto et al .: J. Antib.
iotics, 44, 1286 (1991), T. Hidaka et al .: J. Antibi
otics, 45, 1008 (1992), T. Kuzuyama et al .: J. Antibi
otics, 45, 1812 (1992), T. Kuzuyama et al .: J. Antib
iotics, 46, 1478 (1993))

In addition to fosfomycin as a compound having a C—P bond, there is bialaphos, which is a herbicide produced by Streptomyces hygroscopicus, and its biosynthesis was also conducted by the present inventors. It has been studied in detail and the early steps of biosynthesis have been reported to be similar reactions. (T.Hidak
a et al .: Actinomycetol., 5, 112 (1991), T. Hidaka e
t al .: Actinomycetol., 6, 105 (1992), K. Kamigiri e
t al .: J. Antibiotics, 45, 781 (1992))

[0008] In studies on fosfomycin biosynthesis-related genes, the present inventors showed that biosynthesis-related genes exist in clusters, and the first step of fosfomycin biosynthesis, the phosphoenolpyruvate phosphomutase gene and the second stage. The nucleotide sequence of the phosphonopyruvate decarboxylase gene, which is a stage, has been determined.
(Japanese Patent Application No. 5-282973)

[0009] Further, as a fosfomycin production method currently industrially used, there is a method of chemically epoxidizing cis-propenylphosphonic acid. However, in the chemical synthesis method, racemization occurs and optical resolution is required. The production yield is limited. Therefore, a method for producing fosfomycin using cis-propenylphosphonic acid as a substrate, which pays attention to the structure specificity and selectivity of the biosynthetic reaction of microorganisms, was examined, and a method using a microorganism of the genus Penicillium (RF White et al .: Applied Mic
robiology, 22, 55 (1971)) and methods using microorganisms of the genus Servibrio have been reported. (K. Aisaka et al .: Appl
Microbial Biotechnol 36, 431 (1992))

As described above, fosfomycin can be produced by the epoxidation reaction of microorganisms using cis-propenylphosphonic acid, which is an intermediate raw material of the synthetic method, as a substrate.
The present inventors have reported an interesting finding that Streptomyces actinomycete capable of producing fosfomycin does not produce fosfomycin using cis-propenylphosphonic acid as a substrate. (H.Seto et al .: J. Anti
biotics, 44, 1286 (1991))

That is, it has been shown that actinomycetes capable of producing fosfomycin have a different epoxidation mechanism from Penicillium spp. And Servibrio spp. Sex was suggested. In the biosynthesis of actinomycetes capable of producing fosfomycin, the substrate for the epoxidation reaction is presumed to be 2-hydroxypropylphosphonic acid.

[0012]

One of the problems to be solved by the present invention is to identify a methylation enzyme gene and an epoxidation enzyme gene, which are two important reactions of fosfomycin biosynthesis that have not yet been clarified. The basic knowledge for artificially controlling the enzyme activity by determining the nucleotide sequence is obtained.

The second problem is to prepare a recombinant microorganism transformed with a plasmid containing an epoxidase gene, express the epoxidase, and use 2-hydroxypropylphosphonic acid as a substrate of the epoxidase as a raw material. Is to provide a novel method for producing fosfomycin.

In the fosfomycin production method using a recombinant microorganism transformed with a plasmid containing an epoxidase gene, the expression of the epoxidase gene is controlled and promoted by utilizing a high expression promoter and selecting a microorganism having high transcription ability. It is thought that it is possible to dramatically increase the production of fosfomycin.

[0015]

[Means for Solving the Problems] Genes related to fosfomycin biosynthesis including a methylation enzyme gene and an epoxidation enzyme gene exist in a cluster form, and the gene cluster has already been cloned by the present inventors.
That is, the DNA of the fosfomycin-producing bacterium was partially digested with BamHI, which is a restriction enzyme, to prepare a plasmid inserted into the BamHI site of pIJ922. The Myces vedemorensis NP7 strain was transformed and collected using the restoration of the fosfomycin-producing ability as an index. (Agricultural Chemistry Society of Japan 1993 Conference (Sendai)
(Lecture number 3Ka3, Japanese Patent Application No. 5-282973)

The cloned gene cluster is approximately
The strain of Streptomyces vedemorensis NP7 / pFBG23, which has a size of 23 kb and was transformed with a plasmid (pFBG23) having a gene cluster inserted,
Deposited as RMP-13840 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

The phosphoenolpyruvate phosphomutase gene, which is the first-step enzyme of fosfomycin biosynthesis, and the phosphonopyruvate decarboxylase gene, which is the second-step enzyme, present in the gene cluster have already been identified and their nucleotide sequences have been determined. According to the present invention, a DNA fragment containing a methylase gene that is a third-stage enzyme and an epoxidase gene that is a fourth-stage enzyme is collected, and both genes are specified and the nucleotide sequence is determined. It was

Although it is also possible to use the fosfomycin-producing strain Streptomyces vedemorensis ATCC21239 strain as a DNA donor used for collecting DNA containing the methylating enzyme gene and the epoxidizing enzyme gene and determining the nucleotide sequence, DNA of Streptomyces vedemorensis NP7 / pFBG23 strain, which is a strain transformed with a plasmid (pFBG23) having a gene cluster inserted,
It is efficient to use it as a donor and use a so-called subcloning technique.

Known techniques for gene recombination such as collection of plasmids required for subcloning, cleavage of DNA with restriction enzymes, integration of DNA fragments into vector plasmids and transformation of host strains with the generated hybrid plasmids are known. Examples of the method include Yasutaka Takagi's “Genetic Manipulation Experiment” (Kodansha) and Hopman et al. (Genetic Manipulation of Streptomyces, A lab).
You can refer to the oratory manual (1985)).

Restriction enzymes and ligases are commercially available, and examples of suitable vector plasmids include pIJ702.
(Available from John Innes Laboratories. Reference: J. General Mi.
crobiology, 129, 2703-2714 (1983)), and pIJ680 (John
Available from Innes Laboratories. Literature: DAHopwood et a
l .: Genetic Manipulation of Streptomyces, A labora
tory Manual (1985)) and pIJ922 (available from John Innes Laboratories) Reference: DJ.Lydiate et al .: Gene 35, 223
(1985)) and the like.

The host bacterium used for transformation can be appropriately selected from actinomycetes, Escherichia coli, Bacillus subtilis and other microorganisms depending on the type of vector used. As a preferred example of a host bacterium suitable for the purpose of the present invention, it is possible to list Streptomyces bedemorensis NP7 strain and its mutant strain that have good transformation efficiency and are capable of characterizing recombinants obtained by transformation. it can. Streptomyces bedemorensis NP7 strain has been deposited as FERM P-13838 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

In the case of cloning the epoxidase gene, the expression state of the enzyme can be grasped as the amount of conversion to fosfomycin using 2-hydroxypropylphosphonic acid as a substrate, and therefore it is easy to handle and used in many gene expression experiments. The existing Streptomyces lividans 66 can be used. This microorganism has been internationally deposited as FERM BP-737 at the Institute of Biotechnology, Institute of Industrial Science and Technology.

In order to introduce a vector into which a DNA fragment has been incorporated into a host bacterium, the most efficient method is selected depending on the host bacterium and the type of the vector. The most common method is to transform protoplasts. For selection of recombinants obtained by transformation, a genetic index possessed by the vector used, for example, antibiotic resistance, melanin production ability (MJBibb et al .: Molec. Gen. Genet. 154, 155
(1977), Nature 274, 398 (1978)) and the like can be used.

The plasmid into which the gene cluster containing the DNA fragment containing the methylating enzyme gene and the epoxidizing enzyme gene is inserted, the recombinant microorganism transformed with the plasmid is cultivated, and it is easily isolated from the cultured cells by a known method. Can be separated. A restriction enzyme cleavage map can be prepared by cutting the DNA fragment inserted into the isolated plasmid with various restriction enzymes, and performing agarose gel electrophoresis to analyze the size of each fragment.

FIG. 1 shows a restriction enzyme map of the insert DNA of the plasmid pFBG23 and a DNA fragment containing the epoxidase gene whose nucleotide sequence was determined by the present invention (pFBG51 insert DN
The position of A) is shown.

DNA inserted in the isolated plasmid
In order to confirm that the methylating enzyme gene and the epoxidizing enzyme gene are present in the fragment, the following two methods were used alone or in combination. One is a method of transforming the fosfomycin biosynthetic mutant strain with the plasmid and observing the recovery of the fosfomycin-producing ability of the recombinant, which was obtained by mutating Streptomyces vedemorensis NP-7 strain as a mutant strain. A mutant strain NP-17 strain lacking both the second stage and the third stage was used.

The NP-17 strain is a mutant strain that does not produce fosfomycin without addition or addition of 2-aminoethylphosphonic acid, but produces fosfomycin with addition of 2-hydroxypropylphosphonic acid. The presence of the methyltransferase gene can be confirmed by restoration of the fosfomycin-producing ability. The presence of the epoxidase gene can be confirmed by recovering the fosfomycin-producing ability by adding 2-hydroxypropylphosphonic acid using a mutant strain that lacks both the second step and the fourth step in the same manner. 2-Aminoethylphosphonic acid can be purchased from Sigma and 2-hydroxypropylphosphonic acid was synthesized by the method of Preis et al. (S. Preis et a
l .: J. Am. Chem. Soc. 77, 6225 (1955)) The details of the method are described in Japanese Patent Application No. 5-282973.

In addition, in order to confirm the presence of the epoxidase gene, Streptomyces lividans can be used in addition to the fosfomycin-producing strain or its mutant strain. By transforming the plasmid into these host strains, The expression of the recombinant epoxidase gene can be observed by the production of fosfomycin with the addition of 2-hydroxypropylphosphonic acid.

Expression of the epoxidation enzyme gene was carried out by using a crude enzyme solution obtained by subjecting a microbial culture to ultrasonic treatment to extract 2-
It can also be seen by the production of fosfomycin by an enzymatic reaction using hydroxypropylphosphonic acid as a substrate. The plasmid pFBG51 into which the DNA fragment containing the methylation enzyme gene and the epoxidation enzyme gene was inserted was selected by such a method. Recombinant Streptomyces lividans / pFBG51 obtained by transforming Streptomyces lividans with the plasmid pFBG51 has been deposited at the Institute of Biotechnology, Institute of Industrial Science as FERM P-14243.

The second method is to find a gene sequence, so-called open reading frame (ORF), which is presumed to encode the amino acid sequence of the enzyme protein by determining the nucleotide sequence of the DNA fragment by the method of Wright et al. (F. Wright). et a
l .: Gene 113, 55 (1992)), a method of estimating the characteristics of an enzyme protein encoded by a gene from the homology of an amino acid sequence deduced from a nucleotide sequence by comparing it with a previously published amino acid sequence. .

To determine the nucleotide sequence of a DNA fragment containing the methylation enzyme gene and the epoxidation enzyme gene, plasmid pFBG23 or pFBG51 was used.

The PstI-BamHI fragment shown in FIG.
bo) or pUC118 (Takara) and the like to prepare a plasmid for nucleotide sequence analysis, and further prepare a deletion plasmid and single-stranded DNA by a known method to prepare Ta of Applied Biosystems.
q The nucleotide sequence was determined using the Dye Primer Cycle Sequencing kit and the DNA sequencer ABI 373A.

The nucleotide sequence of the DNA fragment (PstI-BamHI fragment) containing the methylating enzyme gene and the epoxidizing enzyme gene inserted in the plasmid pFBG51 is as shown in the sequence of SEQ ID NO: 1 in the sequence listing described later. Four open reading frames (ORFs) were present in the 5170-bp DNA fragment whose nucleotide sequence was determined. Positions 1546-428 and 2244-1543 in the sequence of SEQ ID NO: 1 in the sequence listing, respectively.
Positions 2612-4216 and 4233-4829 (start codon-stop codon). It is known that biosynthesis genes exist in clusters in many antibiotics.
The two ORFs are also considered to be genes encoding fosfomycin biosynthesis related enzymes.

The amino acid sequence deduced from the base sequence of the ORF was subjected to homology search using the database of SWISS-PROT and compared with about 30,000 sequences. As a result of homology search, it was confirmed that the amino acid sequence encoded by the sequence at position 2612-4216 (including the termination codon) had significant homology with magnesium-protoporphyrin monomethyl ester cyclase. Methylcobalamin is involved in the fosfomycin methylation reaction, and this sequence was presumed to be a methylase gene based on the structural similarity between cobalamin and protoporphyrin.

In a recovery test of the fosfomycin productivity by addition of 2-aminoethylphosphonic acid using the methylation enzyme gene-deficient strain NP-17 strain by subcloning, the DNA fragment containing the sequence of this region contains the methylation enzyme gene. Things were confirmed. Similarly, the sequence of positions 4233-4829 (including the stop codon) could be identified as the epoxidase gene. Thus, the present invention was completed by determining the entire nucleotide sequence of a DNA fragment containing a methylation enzyme gene and an epoxidation enzyme gene, which are important enzymes of fosfomycin biosynthesis, and specifying each enzyme gene.

Also, a recombinant Streptomyces lividans / pFBG51 transformed with a plasmid, for example, pFBG51 containing a DNA fragment containing an epoxidase gene is 2-
Since fosfomycin is produced at a high yield using hydroxypropylphosphonic acid as a substrate, it is possible to produce fosfomycin by a novel production method using 2-hydroxypropylphosphonic acid as a raw material.

In the novel method for producing fosfomycin using 2-hydroxypropylphosphonic acid as a substrate, transformation is carried out with a plasmid into which a DNA fragment containing an epoxidase gene is inserted in addition to Streptomyces lividans / pFBG51 of the present invention. Any recombinant microorganism that is capable of producing fosfomycin using 2-hydroxypropylphosphonic acid as a substrate can be used.

Further, in the novel method for producing fosfomycin using 2-hydroxypropylphosphonic acid as a substrate of the present invention,
The above microorganisms are cultivated under aerobic conditions and the
-Additional culture in which hydroxypropylphosphonic acid is added and the culture is further continued to accumulate fosfomycin is common. That is, the above-mentioned bacteria are cultivated in a medium containing nutrients that can be used by ordinary microorganisms. As the nutrient source, known ones conventionally used for culturing actinomycetes can be used.

For example, glucose, starch syrup, dextrin, starch, molasses, animal / vegetable oil, etc. may be used as the carbon source. As the nitrogen source, soybean meal, wheat germ, corn steep liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea and the like can be used. In addition, if necessary, sodium, potassium,
It is also possible to add calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid, and other inorganic salts. In addition, organic and inorganic substances that help the growth of bacteria and promote the production of fosfomycin from 2-hydroxypropylphosphonic acid can be appropriately added.

As a method of additional culture, a culture method under aerobic conditions, particularly a deep culture method is most suitable. A suitable temperature for culturing is 25 to 30 ° C, but in most cases, culturing is performed at around 28 ° C. The production of fosfomycin using 2-hydroxypropylphosphonic acid as a substrate varies depending on the culture conditions and the amount of substrate added, but its accumulation usually reaches its maximum within 3 to 7 days in both shaking culture and tank culture. . When the accumulated amount of fosfomycin reaches the maximum, the culture is stopped, and the target substance is isolated and purified from the culture medium.

In addition to the addition culture, the method also includes a method of using immobilized cells in which the cultured cells are fixed on a suitable carrier, an epoxidation enzyme extracted from the cultured cells by ultrasonic treatment, French press or the like. Any method can be used as long as fosfomycin production is observed using 2-hydroxypropylphosphonic acid as a substrate, such as an enzymatic method using an extract.

Fosfomycin produced by using 2-hydroxypropylphosphonic acid as a substrate is an acidic water-soluble substance, and can be purified from a culture solution or an enzyme solution according to its properties. Purification method using ion exchange resin such as Dowex 1x2 or Dowex 50W, purification method by adsorbing on alumina or carbon and eluting with a suitable solvent, separation by molecular size using resin such as Sephadex or ultrafiltration membrane The conventional purification method used for purifying an antibiotic from a culture medium, such as the method described above, the method using solvent extraction or crystallization, can be used. For details of such a purification method, for example, the report of L. Chaiet and the like can be referred to. (L.Chaie
t et al .: J. Antibiotics, 23, 336 (1970))

In the assay of fosfomycin, biological assay methods, silica gel thin layer chromatography and HP
Conventional assay methods used for detecting antibiotics such as chemical assay methods by LC can be used. Examples of the present invention will be shown below, but these are merely examples and do not limit the present invention. It goes without saying that many modified or modified means not exemplified here can be used.

[0042]

Example 1 Methylase gene and epoxidase gene
Method for collecting plasmid having inserted DNA fragment containing gene
For example, pFBG51 is as follows. Straight
Transformation with P. lividans as host bacterium
Bibb et al. (M.J.Bibb et al .: Nature, 274, 3
98 (1978)) by a modified method. That is,
Treptomyces lividans with glycine concentration of 0.5%
100 ml GPY medium (glucose 1.0%, polypeptone 0.5
%, Yeast extract 0.4%, MgSO_Four・ 7H₂O0.05%, K₂
HPO_Four0.1%) was inoculated into a Sakaguchi flask and dispensed at 30 ° C for 24 hours.
After shaking culture for 1 hour, collect mycelium by centrifugation at 10,000 g
After washing once with 0.35M sucrose solution, lysozyme (raw
20 ml of P medium (12% sucrose, manufactured by Kagaku Kogyo Co., Ltd.) (1 mg / ml),
1.4mM K₂SO_Four, 10mM MgCl₂, 0.4mM KH₂PO_Four, 25mM CaCl₂,
 25 mM TES buffer (pH 7.2), 1/500 volume trace element solution)
Made cloudy. (Composition of trace element solution in 1 liter: 40 mg Z
nCl₂, 200mg FeCl₃・ 6H₂O, 10mg CuCl₂・ 2H₂O, 10mg MnCl
₂・ 4H₂O, 10mg Na₂B_FourO₇・ 10H ₂O, 10mg (NH4)₆Mo₇O_{twenty four}・ 4H
₂O) Incubate at 30 ℃ for 30 minutes to form protoplasts.
The hyphae that did not become protoplasts were removed by cotton filtration.
It was Collect protoplasts by centrifugation and in P medium
It was washed once and then suspended in 1 ml of P medium.

To 100 μl of the protoplast solution was added 10 μl of various plasmid DNA (plasmid cleaved pFBG23 with various restriction enzymes and inserted into vector plasmid pIJ702 or pIJ680) solutions, and immediately, 500 μl of P medium containing 25% polyethylene glycol 1000 Was added and the DNA was incorporated by pipetting. After standing for several minutes, 5 ml of P medium was added to terminate the reaction, protoplasts were collected by centrifugation, suspended in 10 ml of R2YE medium containing 0.6% agar, and 2 ml of each was spread on R2YE medium. (0.3M sucrose,
1.4mM K ₂ SO ₄ , 50mM MgCl ₂ , 1% glucose, 0.01% casamino acid, 1/50 amount trace element solution, 0.4mM KH ₂ PO ₄ , 20mM CaC
l ₂ , 0.3% proline, 25 mM TES buffer (pH7.2), 0.5% yeast extract, 2.2% agar) After culturing at 30 ° C for 6 hours, 1 ml of thiostrepton solution (200 μg / ml) was overlaid, and further After culturing for 3 days, a recombinant was obtained.

An agar piece medium (starch 0.4%, sungrain 0.5%, wheat germ 0.2%, K ₂ HPO ₄ 0.01%, CoCl ₂ .6H) was prepared by using the obtained recombinant to confirm the production of fosfomycin.
2-hydroxypropylphosphonic acid was added to a medium having a composition of ₂ O 0.0001% and agar 2.2% at 100 μg / ml) and cultured for 5 days at 27 ° C., then fosfomycin from 2-hydroxypropylphosphonic acid Productivity was determined by the presence or absence of an inhibition circle for Proteus bulgaris. It is shown that the epoxidase gene is present and expressed in the recombinant plasmid capable of producing fosfomycin.

One of the recombinants thus obtained, Streptomyces lividans / pFBG51, was cultured, and the plasmid was extracted from the cultured cells by a known method.
In the same manner, various mutants of Streptomyces vedemorensis NP7 strain were transformed with this plasmid, and the ability to produce fosfomycin from 2-aminoethylphosphonic acid and 2-hydroxypropylphosphonic acid in the recombinant was investigated and the epoxidase gene The presence of both the gene and the methylase gene was confirmed.

[0046]

[Example 2] Streptomyces lividans / pFBG51
Preculture medium (starch 2.0%, peptone 1.0%, meat extract
0.3%, K ₂ HPO ₄ 0.05%, pH 7.0) and incubate with shaking at 28 ° C for 24 hours, and then fosfomycin production medium (starch 4.
0%, sun grain 5.0%, wheat germ 2.0%, K ₂ HPO ₄ 0.1%, C
It was transplanted to oCl ₂ .6H ₂ O 0.0001%, pH 8.0). 2-Hydroxypropylphosphonic acid was added to the production medium at 300 μg / ml, and shake culture was carried out at 28 ° C. for 4 days. The amount of fosfomycin produced in the culture filtrate was about 100 μg / ml in a bioassay using Proteus bulgaris MB-838.

[0047]

Example 3 The inserted DNA fragment of the plasmid pFBG51 containing the methylation enzyme gene and the epoxidation enzyme gene was

As shown in Figure 1, the approximately 5.1 kb PstI-BamH in pFBG23.
Shown as I fragment. This PstI-BamHI fragment was transformed into pTZ18
And pTZ19 (Toyobo) were cloned into BamHI and PstI, respectively, and the deletion plasmid using the restriction enzyme site from these plasmids and the deletion plasmid using the deletion kit for kilo-Sequence (Takara Shuzo) according to the attached instructions Prepared.

Escherichia coli JM109 strain transformed with these deletion plasmids was transformed into LB medium containing 50 μg / ml of ampicillin (B
acto trypton 1%, Bacto yeast extract 0.5%, NaCl 0,
After pre-culturing with 5% pH 7.0, 2xYT medium (Bacto trypton 1.6
%, Bacto yeast extract 1.0%, NaCl 0,5% pH 7.0), and cultured for 1 hour at 37 ° C., after which phage M13KO7 was added and further cultured for 7 to 8 hours. Single-stranded DNA was prepared from the culture supernatant and used for determining the nucleotide sequence.

Nucleotide sequence determination was performed by Applied Biosystems.
It was performed using Taq Dye Primer Cycle Sequencing kit and DNA sequencer ABI 373A. As a result, the base sequence of 5170 base was determined. The determined nucleotide sequence is as shown in the sequence of SEQ ID NO: 1 in the sequence listing described below. The open reading frame (ORF) was examined based on the determined nucleotide sequence. Bibb et al. Report (Gene, 30,
157 (1984)) and Wright et al. (F. Wright et al .: Gene
113, 55 (1992)), four ORFs were found in the sequence of SEQ ID NO: 1 in the sequence listing. 1546-428th, 2244-1543th, and 2nd in the sequence of SEQ ID NO: 1 in the sequence listing, respectively.
It is located at positions 612-4216 and 4233-4829 (start codon-stop codon).

From the result of subcloning and the result of homology search, the nucleotide sequence of 2612-4216 position (including stop codon) is methylation enzyme gene, and the nucleotide sequence of 4233-4829 position (including stop codon) is epoxidation enzyme. Identified as a gene. The methylase gene starts from the ATG at the 2612th position in the sequence listing SEQ ID NO: 1 and ends at the TGA at the 4214th position. The amino acid sequence corresponding to the base sequence was also determined. The sequence is shown as an amino acid sequence corresponding to positions 2612-4213 in the sequence of SEQ ID NO: 1 in the sequence listing, and the same amino acid sequence is shown as the sequence of SEQ ID NO: 2.

The epoxidase gene starts from the ATG at the 4233th position and ends at the TGA at the 4827th position in the sequence listing. The amino acid sequence corresponding to the base sequence was also determined. The sequence is shown as the amino acid sequence corresponding to positions 4233-4826 in the sequence of SEQ ID NO: 1 in the sequence listing, and the same amino acid sequence is shown as the sequence of SEQ ID NO: 3.

The role of the two ORFs located at positions 1546-428 and 2244-1543 is currently unknown, but biosynthesis-related genes may exist in clusters in many antibiotics. Known and these two ORs
F is also considered to be a fosfomycin biosynthesis-related gene

[0053]

[Sequence list]

SEQ ID NO: 1 Sequence length: 5170 Sequence type: Nucleic acid Number of strands: Double-strand Topology: Linear Sequence type: Genomic DNA Origin Biological name: Streptomyces bedemolensis (Strept
omyces wedmorensis) Strain name: ATCC21239 strain Sequence features Characteristic symbol: mat peptide Location: 2612..4213 4233..4826 Method of determining features: E sequence CTGCAGGACG AGCAGGGGGA TCGCGAGGATCGCGAGCCCG TCGCCGATCG 120 CGGGGCGAGT GCCACTCAGG CCACCGCCTC GACGAGCGCG CCGACGATCT GGCAGGCCTG 180 GCTCATGGTC AGCGTCGCGA ACCCCTCGGG CTCGACCGGC TCCCCGGCGG CCAGCGGCCG 240 GCCGTCCGGC GACAGCCGGT CGCCCCACCT GATCTGCCGG CGGAAGGCCG CGGACAGCTC 300 CGCGCCCTCG GCCGCCAGCC ACTCGGCCAC GACCCGCTGG ATCTCGGGGG CCGTCCGGTC 360 CACGCGCTCG GGCCAGGCCG CGATGGCCGT ACGGACCCGG GCGAGCAGGT CCCCGACCAG 420 TGCCACGTCA CGCGGCTCGC GGGAGGACGG CCACAGCAGG AAGGGCGCGG TGGGTGCGAT 480 GACGAACAGC AGGGGGAGCG GCCCAGGGGA GGTCCCGCGC CACGGCCTCG TACATCCACT 540 TGGTCTTCTT CGCGCCGCTG AAGAAGTACC CGCGGCGCTG CGGCGGGTAG CCGCCCATCG 600 CCAGGGCGCC CCACTGCACG TGCACGTCGA AGTCGCCGCA GACCGGGGGC TGTCCGGGCG 660 CGCGGTGGGT GCCGAGGACC GCGTAGAGAA GGGTGACCGC GTCCACCGAC AG CAGCGGCG 720 CGTGCGGCGC CGGGGCCGCG TCCTGGCCGG TGAACCGCCC GGCGGCGACC GCCGCCCGCG 780 CGGGCAGCAG GCTCGCCGCG ACCAGCGCGT CGGCCGTCGG GTAGTCGAAG GGCCCCAGGT 840 GGTAGCCCGA AGCGTCGGCC GTCGGCTTGA ACCGCTCCAC GCTTCCGTCG CGGTGCACGA 900 TCCGGTCGAA CACGTAGGAC AGGGCCCGGG TGTCCGTCCC GGCGGCCCGG AGCAGCGTGT 960 GCAGCACCTC CGTGACCCGC TCCTCCTCGC CCGTCAGGAG GTCGGAGTGG AACGTGGCGT 1020 CGGGGAAGTG CTCCCGCATC CGGCGGCGGT GCAGCTCGGT GAGGGGCAGT CGTTCGCCCT 1080 CCCACTCGGT CTCCACGTAC GGCACGAACA GCGATCCCTC GCCGTCGTGC CGCTCCATGC 1140 CCGCCGGCGA CCAGTGCCGG AGGGGGAGCC TGCTGCGTAC GCCGGCCTCG ACCAGGGAGC 1200 CGGGCAGGAA GAGGGTGTCA CCGCTCGCCA GGGCGGTGTC CTCGGTCTGG TTGAAGTCGT 1260 AGAGCAGGGG GCGGGCGAGG CCCGGAACGC CGCAGACCAC CCGGTTGTAG TCCAGGACCG 1320 TGCTGCGTCC GGAACCGATG TCGCCCCAGT CGGCGAGGTG CCCGGTGCGA ATGGGGGTGA 1380 GCGGCAACGC GGCGATCGAG GCGGTCAGTT CGCCCATCGG GAGCTGTACG CGTCCCATGT 1440 GGCGGGCGAC GAACCCCGAG CGTCCCGACC GGAGTTCGGC CCACGATTCC TCGTCGAGGC 1500 CGATCGCCTT GAGATCCTGT GGTGCCAGGC CGAGCTCCGA GGTCATCGGG AAGCGGCCAT 15 60 GTCCAGGACG GCCCGGCCCA GGATGCGGGT CACGTCGTAC ACGTGCGGGG GGAGCTCGGG 1620 CAGCAGCGGC GACAGGTCCG TGCAGCCGAC GACGACGCCG TCCCCGGGCC GCGTCCACTC 1680 CCCTTCGACG GCCTTGCGCA ACTGGGCCGA AGCCTCCTGC ACACGGCCGG ACTTGACCAT 1740 CTCGACGCAT TCCTGGATCT CGCGCTGCGT GGCGGGGTCC GGGTAGACGG GAGTGAGGCC 1800 GGCGTCCACC AGGGCCCGGT GCAGCAGACC GAGCTTCAGT GCGCCGTCGG TGACCGCGAG 1860 GGCGGGGCGC AGGACTCCGG CCTCCCGGAG GCCGGCGGCC ACGACCCGGA GCATGTCGAC 1920 CACGGGAACG TCCACCGCGG CCGAGACCTC GTCGTAGTAG GCCTGCGTGG TGACCGACGG 1980 CATCGCGATC AGCTGGGCGC CGGCGTCCTG GAGACGCCGC GCCACGGCCT GGAGCTGTTC 2040 CACCGGTGAG GGGCCGTCGG ACAGGAGGTG GTCGAGGCGA CTGGGGATGG CGGGGCTGGA 2100 GAGGAGGAGG ACCTCGGGGT GCTCCTGGTC GCTGCCGGCC GTCGTCTGCT CGACGAGACG 2160 CATGTAGAAA TTAGCGCAGG CAAGGGGTCC AAGCCCGCCG ATAATGCCTA TCTTCTGCCA 2220 GTGGTCTATC GCGGCGTTAC TCACGTAGTT CACCCTGCTT CGGGCGGCGT CCGGAAAGCA 2280 ACCCCGAACC GATGGCATAT GACGGTCATG GCGCATAAGT GTCAACCATG AAGCGGTGCA 2340 AACCGGGCAT CCGTCAGGTC TTGTTGCAAG AATGTTGACG TCTTTCAGAC CTGAGTATTA 2400 CGT TGATCGC CACTTCACGC TCGACGCCGG GGGAACGGCG TAATGGGGCG CACCGAATCC 2460 TTGTCCTTGA TGGTGCAGCG GCTACTGGTT GGGCACGTCC GAACACTCGG GCGGCGGCGT 2520 TTTCACCGGC ATTGGAGAGT GGTATCTCCA TGCGTTCATC CGGGCCCATT TCACGAGAAA 2580 CGCGGCGGTT TTGAGCACTA AACAGAATCT C ATG ACG ATC GGT TCT CTG GGC 2632 Met Thr Ile Gly Ser Leu Gly 1 5 TCC ACC GAG TTC GCC CTG CAC GGG AAG CCG GCC ATC AGA TGG GGC GAT 2680 Ser Thr Glu Phe Ala Leu His Gly Lys Pro Ala Ile Arg Trp Gly Asp 10 15 20 CTG CCC CAG CGC GTC GGA AAG CCG GAG ACG CGG CGC TAC CAG AAG GTG 2728 Leu Pro Gln Arg Val Gly Lys Pro Glu Thr Arg Arg Tyr Gln Lys Val 25 30 35 CTG CTG CTG AAT CCC TCG GCA ACG CTC TTC CGG CAC GAC CTT CCG CGA 2776 Leu Leu Leu Asn Pro Ser Ala Thr Leu Phe Arg His Asp Leu Pro Arg 40 45 50 55 TGT ACG TAC CCC CTC GGC CTG GGG TAC ATC GCG GCC GTC CTC GAG AAG 2824 Cys Thr Tyr Pro Leu Gly Leu Gly Tyr Ile Ala Ala Val Leu Glu Lys 60 65 70 TAC GGC TAC GAG GTC AAG ATC CTC GAC GTC TTC GCG GAG GGC TAC TAC 2872 Tyr Gly Tyr Glu Val Lys Ile Leu Asp Val Phe Ala Gl u Gly Tyr Tyr 75 80 85 AAC GCC CAG CCC GTC GAC GGC GAC GAC CAG TTC CTC CGT TAC GGC CTG 2920 Asn Ala Gln Pro Val Asp Gly Asp Asp Gln Phe Leu Arg Tyr Gly Leu 90 95 100 TCG GAC GAC GAC ATC GTC AAG GTG ATG AAG GAG TTC GGG CCC GAC GTC 2968 Ser Asp Asp Asp Ile Val Lys Val Met Lys Glu Phe Gly Pro Asp Val 105 110 115 GTC GGC ATC TCG AGC ATC TTC AGC AAC CAG GCC GAC AAC GTG CAC CAC 3016 Val Gly Ile Ser Ser Ile Phe Ser Asn Gln Ala Asp Asn Val His His 120 125 130 135 CTT CTG AAG CTG GCG GAC CTC GTC ACG CCC GAG GCC GTC ACC GCG ATC 3064 Leu Leu Lys Leu Ala Asp Leu Val Thr Pro Glu Ala Val Thr Ala Ile 140 145 150 GGC GGA GCG CAC GCG CGC TAC TTC CCG AAG GCC TGT CTG GAC GAC CCG 3112 Gly Gly Ala His Ala Arg Tyr Phe Pro Lys Ala Cys Leu Asp Asp Pro 155 160 165 AAC CTC GAC GCG GTG TTC CTC GGC GAA GGC GAG ATG ACC TTC CTG CTG 3160 Asn Leu Asp Ala Val Phe Leu Gly Glu Gly Glu Met Thr Phe Leu Leu 170 175 180 TGG ATG GAG CAC CTC AAC GGA AAC GTG AGC GAC GAC GAG GTC CAC GGC 3208 Trp Met Glu His Leu Asn Gly Asn Val Ser Asp Asp Glu Val His Gly 185 190 195 ATC GCG TGG CGT GAC CGC GAC GGC AAG GTC CAG ATC AAG CCC GAG CTG 3256 Ile Ala Trp Arg Asp Arg Asp Gly Lys Val Gln Ile Lys Pro Glu Leu 200 205 210 215 CCG CTG ATC AGC TCG ATG CGC CCC GAA GGC CCC GAG ACG GGC AAG TCC 3304 Pro Leu Ile Ser Ser Met Arg Pro Glu Gly Pro Glu Thr Gly Lys Ser 220 225 230 TCT CCC ATG CTG AGC ATG GCC GGC GAA CTG GAC CAC ATC CCG TTC CCC 3352 Ser Pro Met Leu Ser Met Ala Gly Glu Leu Asp His Ile Pro Phe Pro 235 240 245 GCC TGG CAC CAC TAC AAC ATG GAG AAG TAC TTC GAG ATC AAG GCC TAC 3400 Ala Trp His His Tyr Asn Met Glu Lys Tyr Phe Glu Ile Lys Ala Tyr 250 255 260 CAG TCG CCG TAC ACG GTC GGC TCC CGG GTG GGC CAG CTC TAC ACC AGC 3448 Gln Ser Pro Tyr Thr Val Gly Ser Arg Val Gly Gln Leu Tyr Thr Ser 265 270 275 CGC GGC TGC ACG GCC CAC TGC ACC TTC TGC ACG ACC ACC CAC TTC TGG 3496 Arg Gly Cys Thr Ala His Cys Thr Phe Cys Thr Thr Thr His Phe Trp 280 285 290 295 GGT CAG AAG CTC CGC CGG CGC AGC GTC CAG GAC GTG GTC GAC GAG GTC 3544 Gly Gln Lys L eu Arg Arg Arg Ser Val Gln Asp Val Val Asp Glu Val 300 305 310 CTG AGG CTG CGC GAC GAA TAC GGA ATC GAC GAA TTC CAT ATT CAG GAC 3592 Leu Arg Leu Arg Asp Glu Tyr Gly Ile Asp Glu Phe His Ile Gln Asp 315 320 325 GAC AAC ATC ACG AAC GAC ATG GAC CAC GCC CGC GAG CTG TTC CGC GCG 3640 Asp Asn Ile Thr Asn Asp Met Asp His Ala Arg Glu Leu Phe Arg Ala 330 335 340 TTC AAG GAG GTC GGC CTC CCC TGG GCG ACC CCG CAG GGC ACC GCC CTG 3688 Phe Lys Glu Val Gly Leu Pro Trp Ala Thr Pro Gln Gly Thr Ala Leu 345 350 355 TGG CGC ATG GAC GAA GAG CTG CTC GAC CTC ATG GCG GAG TCG GGC GCC 3736 Trp Arg Met Asp Glu Glu Leu Leu Asp Leu Met Ala Glu Ser Gly Ala 360 365 370 375 TAC CAG GTG ACG TTC GCC ATC GAG AGC GGC GTG CAG CGA GTC CTC AAG 3784 Tyr Gln Val Thr Phe Ala Ile Glu Ser Gly Val Gln Arg Val Leu Lys 380 385 390 GAA CTC ATC AAG AAG CCG CTC AAC CTC GAA CGG ACC TCG CAC CTC ATC 3832 Glu Leu Ile Lys Lys Pro Leu Asn Leu Glu Arg Thr Ser His Leu Ile 395 400 405 AAG TAC GCG AGA AGC CTT GGA ATG CAC GTC CAC GGC TTC TTC ATC ATC 3880 Lys Tyr Ala Arg Ser Leu Gly Met His Val His Gly Phe Phe Ile Ile 410 415 420 GGC ATG CCG CCG ATG TGC GGA AAC GCC GGC GAA AGC ATC GAG GAG ATG 3928 Gly Met Pro Pro Met Cys Gly Asn Ala Gly Glu Ser Ile Glu Glu Met 425 430 435 CAG GCG TCG TAC GAC TAT GCC GAA GAA GCC GGA TTC AGC AGC GCT TCC 3976 Gln Ala Ser Tyr Asp Tyr Ala Glu Glu Ala Gly Phe Ser Ser Ala Ser 440 445 450 455 TTC TTC GCG GCA TCG CCG ATC GTC GGT TCC GAG CTC CTG CGC GAG TGC 4024 Phe Phe Ala Ala Ser Pro Ile Val Gly Ser Glu Leu Leu Arg Glu Cys 460 465 470 ATT CGC CAG GGG TTC GTG GAC CCG GAG GAG TCT CTT TAC CGC ATG ACC 4072 Ile Arg Gln Gly Phe Val Asp Pro Glu Glu Ser Leu Tyr Arg Met Thr 475 480 485 TAC AAG CAG GGG ATC ATC AAC GTC CCC GGC CTG TGG GAC GGC GAG GAG 4120 Tyr Lys Gln Gly Ile Ile Asn Val Pro Gly Leu Trp Asp Gly Glu Glu 490 495 500 ATC GCG GAG CTC GCC GCG AAA TTC AAC CGG GAC TTC AAT GCG CGG CGC 4168 Ile Ala Glu Leu Ala Ala Lys Phe Asn Arg Asp Phe Asn Ala Arg Arg 505 510 515 GAC CGG GCT TAC ACG CCG CAG AAG CAG TGG AAC GCA AAC CAG TAC TGA 4216 Asp Arg Ala Tyr Thr Pro Gln Lys Gln Trp Asn Ala Asn Gln Tyr *** 520 525 530 534 AATC TGGGAGGGCG AA ATG AGC AAC ACC AAG ACG GCG AGC ACG GGC 4262 Met Ser Asn Thr Lys Thr Ala Ser Thr Gly 1 5 10 TTC GCG GAG CTC CTC AAG GAC CGG CGC GAG CAG GTC AAG ATG GAC CAC 4310 Phe Ala Glu Leu Leu Lys Asp Arg Arg Glu Gln Val Lys Met Asp His 15 20 25 GCC GCC CTT GCC TCG CTC CTC GGG GAG ACC CCC GAG ACG GTG GCG GCC 4358 Ala Ala Leu Ala Ser Leu Leu Gly Glu Thr Pro Glu Thr Val Ala Ala 30 35 40 TGG GAG AAC GGC GAG GGC GGC GAG CTG ACG CTC ACG CAG CTG GGC AGG 4406 Trp Glu Asn Gly Glu Gly Gly Glu Leu Thr Leu Thr Gln Leu Gly Arg 45 50 55 ATC GCC CAC GTG CTC GGC ACG TCG ATC GGC GCC CTC ACC CCG CCG GCG 4454 Ile Ala His Val Leu Gly Thr Ser Ile Gly Ala Leu Thr Pro Pro Ala 60 65 70 GGC AAC GAC CTC GAC GAC GGC GTC ATC ATC CAG ATG CCC GAC GAG CGC 4502 Gly Asn Asp Leu Asp Asp Gly Val Ile Ile Gln Met Pro Asp Glu Arg 75 80 85 90 CCG ATC CTC AAG GGT GTG CGG GAC AAC GTC GAC TAC TAC GTC TAC AAC 4550 Pr o Ile Leu Lys Gly Val Arg Asp Asn Val Asp Tyr Tyr Val Tyr Asn 95 100 105 TGT CTC GTC CGC ACC AAG CGT GCG CCT TCG CTC GTC CCC CTC GTG GTG 4598 Cys Leu Val Arg Thr Lys Arg Ala Pro Ser Leu Val Pro Leu Val Val 110 115 120 GAC GTG CTG ACG GAC AAC CCC GAC GAC GCG AAG TTC AAC TCG GGC CAC 4646 Asp Val Leu Thr Asp Asn Pro Asp Asp Ala Lys Phe Asn Ser Gly His 125 130 135 GCC GGC AAC GAG TTC CTC TTC GTG CTC GAG GGC GAG ATC CAC ATG AAG 4694 Ala Gly Asn Glu Phe Leu Phe Val Leu Glu Gly Glu Ile His Met Lys 140 145 150 TGG GGC GAC AAG GAG AAC CCG AAG GAG GCC CTC CTC CCG ACC GGC GCG 4742 Trp Gly Asp Lys Glu Asn Pro Lys Glu Ala Leu Leu Pro Thr Gly Ala 155 160 165 170 AGC ATG TTC GTG GAG GAG CAC GTG CCG CAC GCC TTC ACG GCG GCC AAG 4790 Ser Met Phe Val Glu Glu His Val Pro His Ala Phe Thr Ala Ala Lys 175 180 185 GGC ACG GGT TCC GCG AAG CTG ATC GCC GTC AAC TTC TGA C CCACGGAGT 4840 Gly Thr Gly Ser Ala Lys Leu Ile Ala Val Asn Phe *** 190 195 198 ACGGGTGGAC TCCCTCCACC CGTACCTCGC GGTCCCGTGA GGCAGCCCGG TCTGCCGAGT 4900 TGCCGAATGG ACGAGACAGG ACGATGGCAG AAGTCGCCTC CCAGGAAACC GAGTTCGCGG 4960 CCTTCGCGTT CGGGTCGGTC GTCGAGCGCC GGGACGAACT GGAGGGGCGG CCGTGGATCT 5020 CGTATCCCGT CCGCGTGGTG GCCGACACCC CCGAGCTCGT CGCCGTCTAC CTGTCCCACG 5080 GCACCCTCCT CACCTTCGGA GACGGCCCCT TCAGCTGGGG CCCGCACCCC TGGGGACCGT 5140 TCGGCGACCG GTGGCAGAGC GCCGGGATCC 5170

[0054]

[Sequence list]

 SEQ ID NO: 2 Sequence length: 534 Sequence type: Amino acid Topology: Sequence type: Protein Sequence feature symbol: mat peptide Method for determining feature: E sequence Met Thr Ile Gly Ser Leu Gly Ser Thr Glu Phe Ala Leu His Gly Lys 1 5 10 15 Pro Ala Ile Arg Trp Gly Asp Leu Pro Gln Arg Val Gly Lys Pro Glu 20 25 30 Thr Arg Arg Tyr Gln Lys Val Leu Leu Leu Asn Pro Ser Ala Thr Leu 35 40 45 Phe Arg His Asp Leu Pro Arg Cys Thr Tyr Pro Leu Gly Leu Gly Tyr 50 55 60 Ile Ala Ala Val Leu Glu Lys Tyr Gly Tyr Glu Val Lys Ile Leu Asp 65 70 75 80 Val Phe Ala Glu Gly Tyr Tyr Asn Ala Gln Pro Val Asp Gly Asp Asp 85 90 95 Gln Phe Leu Arg Tyr Gly Leu Ser Asp Asp Asp Ile Val Lys Val Met 100 105 110 Lys Glu Phe Gly Pro Asp Val Val Gly Ile Ser Ser Ile Phe Ser Asn 115 120 125 Gln Ala Asp Asn Val His His Leu Leu Lys Leu Ala Asp Leu Val Thr 130 135 140 Pro Glu Ala Val Thr Ala Ile Gly Gly Ala His Ala Arg Tyr Phe Pro 145 150 155 160 Lys Ala Cys Leu Asp Asp Pro Asn Leu Asp Ala Val Phe Leu Gly Glu 165 170 175 Gly Glu Met Thr Phe Leu Leu Trp Met Glu His Leu Asn Gly Asn Val 180 185 190 Ser Asp Asp Glu Val His Gly Ile Ala Trp Arg Asp Arg Asp Gly Lys 195 200 205 Val Gln Ile Lys Pro Glu Leu Pro Leu Ile Ser Ser Met Arg Pro Glu 210 215 220 Gly Pro Glu Thr Gly Lys Ser Ser Pro Met Leu Ser Met Ala Gly Glu 225 230 235 240 Leu Asp His Ile Pro Phe Pro Ala Trp His His Tyr Asn Met Glu Lys 245 250 255 Tyr Phe Glu Ile Lys Ala Tyr Gln Ser Pro Tyr Thr Val Gly Ser Arg 260 265 270 Val Gly Gln Leu Tyr Thr Ser Arg Gly Cys Thr Ala His Cys Thr Phe 275 280 285 Cys Thr Thr Thr His Phe Trp Gly Gln Lys Leu Arg Arg Arg Ser Val 290 295 300 Gln Asp Val Val Asp Glu Val Leu Arg Leu Arg Asp Glu Tyr Gly Ile 305 310 315 320 Asp Glu Phe His Ile Gln Asp Asp Asn Ile Thr Asn Asp Met Asp His 325 330 335 Ala Arg Glu Leu Phe Arg Ala Phe Lys Glu Val Gly Leu Pro Trp Ala 340 345 350 Thr Pro Gln Gly Thr Ala Leu Trp Arg Met Asp Glu Glu Leu Leu Asp 355 360 365 Leu Met Ala Glu Ser Gly Ala Tyr Gln Va l Thr Phe Ala Ile Glu Ser 370 375 380 Gly Val Gln Arg Val Leu Lys Glu Leu Ile Lys Lys Pro Leu Asn Leu 385 390 395 400 Glu Arg Thr Ser His Leu Ile Lys Tyr Ala Arg Ser Leu Gly Met His 405 410 415 Val His Gly Phe Phe Ile Ile Gly Met Pro Pro Met Cys Gly Asn Ala 420 425 430 Gly Glu Ser Ile Glu Glu Met Gln Ala Ser Tyr Asp Tyr Ala Glu Glu 435 440 445 Ala Gly Phe Ser Ser Ala Ser Phe Phe Ala Ala Ser Pro Ile Val Gly 450 455 460 Ser Glu Leu Leu Arg Glu Cys Ile Arg Gln Gly Phe Val Asp Pro Glu 465 470 475 480 Glu Ser Leu Tyr Arg Met Thr Tyr Lys Gln Gly Ile Ile Asn Val Pro 485 490 495 Gly Leu Trp Asp Gly Glu Glu Ile Ala Glu Leu Ala Ala Lys Phe Asn 500 505 510 Arg Asp Phe Asn Ala Arg Arg Asp Arg Ala Tyr Thr Pro Gln Lys Gln 515 520 525 Trp Asn Ala Asn Gln Tyr 530 534

[0055]

[Sequence list]

 SEQ ID NO: 3 Sequence length: 198 Sequence type: Amino acid Topology: Sequence type: Protein Sequence feature symbol: mat peptide Characterization method: E sequence Met Ser Asn Thr Lys Thr Ala Ser Thr Gly Phe Ala Glu Leu Leu Lys 1 5 10 15 Asp Arg Arg Glu Gln Val Lys Met Asp His Ala Ala Leu Ala Ser Leu 20 25 30 Leu Gly Glu Thr Pro Glu Thr Val Ala Ala Trp Glu Asn Gly Glu Gly 35 40 45 Gly Glu Leu Thr Leu Thr Gln Leu Gly Arg Ile Ala His Val Leu Gly 50 55 60 Thr Ser Ile Gly Ala Leu Thr Pro Pro Ala Gly Asn Asp Leu Asp Asp 65 70 75 80 Gly Val Ile Ile Gln Met Pro Asp Glu Arg Pro Ile Leu Lys Gly Val 85 90 95 Arg Asp Asn Val Asp Tyr Tyr Val Tyr Asn Cys Leu Val Arg Thr Lys 100 105 110 Arg Ala Pro Ser Leu Val Pro Leu Val Val Asp Val Leu Thr Asp Asn 115 120 125 Pro Asp Asp Ala Lys Phe Asn Ser Gly His Ala Gly Asn Glu Phe Leu 130 135 140 Phe Val Leu Glu Gly Glu Ile His Met Lys Trp Gly Asp Lys Glu Asn 145 150 155 160 Pro Lys Glu Ala Leu Leu Pro Thr Gl y Ala Ser Met Phe Val Glu Glu 165 170 175 His Val Pro His Ala Phe Thr Ala Ala Lys Gly Thr Gly Ser Ala Lys 180 185 190 Leu Ile Ala Val Asn Phe 195 198

[Brief description of drawings]

FIG. 1 is a DNA fragment containing an epoxidase gene whose nucleotide sequence has been determined by the present invention (pFBG51 insert fragment DNA).
Is shown in the restriction map of the pFBG23 insert DNA.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI technical display location C12R 1: 465) (C12P 1/04 C12R 1: 465) (72) Inventor Tomohisa Kuzuyama Tokyo Metropolitan Bunkyo 1-1-1 Yayoi-ku, Institute for Molecular Biology, University of Tokyo (72) Inventor Naomi Takei 1-1-1, Yayoi, Bunkyo, Tokyo Inside, Institute for Molecular Biology, University of Tokyo (72) Haruo Seto Tokyo 1-1-1 Yayoi, Bunkyo-ku, Tokyo Inside Institute for Molecular Biology, University of Tokyo

Claims (9)

[Claims] 1. A DNA comprising a methylating enzyme gene encoding the amino acid sequence represented by the sequence of SEQ ID NO: 2 in the sequence listing or its equivalent. 2. The DNA according to claim 1, wherein the methylase gene is represented by the nucleotide sequence at position 2612-4213 in the sequence of SEQ ID NO: 1 in the sequence listing. 3. A DNA comprising an epoxidase gene encoding the amino acid sequence represented by the sequence of SEQ ID NO: 3 in the sequence listing or its equivalent. 4. The DNA according to claim 3, wherein the epoxidase gene is represented by the nucleotide sequence at position 4233-4826 in the sequence of SEQ ID NO: 1 in the sequence listing. 5. A gene cluster containing an epoxidase gene, and a plasmid containing the gene cluster. 6. A recombinant transformed with the plasmid according to claim 5. 7. The recombinant according to claim 6, wherein the host used for transformation is Streptomyces lividans, which is a microorganism that originally cannot produce fosfomycin. 8. A novel method for producing fosfomycin using 2-hydroxypropylphosphonic acid as a substrate, which uses a recombinant transformed with a plasmid containing an epoxidase gene. 9. The method according to claim 8, wherein the recombinant is Streptomyces lividans / pFBG51.