JPH012585A - DNA containing cytochrome P-450 gene - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to the isolation of DNA containing a gene part that is present in the genus Streptomyces and is involved in the production of cytochrome P-450, which has hydroxylase activity, and its use. . More specifically, ML-2 exists in the genus Stregutomyces.
Sodium 36B (hereinafter referred to as "Na-236B Na J") was converted into sodium 6β-hydroxy-236B (hereinafter referred to as "Na J").
Regarding the production of a protein containing cytochrome P-450 that has a hydroxylase activity that converts it into "C8-514" (hereinafter referred to as "C8-514"), a DNA consisting of approximately 7.1 kb is produced by partial digestion of the restriction enzyme Mbol. The present invention relates to a DNA characterized in that it is present in a fragment and contains a cytochrome P-450 gene having hydroxylase activity.

BACKGROUND OF THE INVENTION DNA recombination experiments using microorganisms have been developed particularly in Escherichia coli, Bacillus subtilis, and yeast. In particular, the development of DNA recombination experiments using Escherichia coli has been remarkable, and it has now been applied not only to the analysis of various genes but also to the industrial production of certain useful peptides. On the other hand, actinomycetes have long been considered important in the F1 fermentation industry because they have a wide variety of abilities in producing secondary foreground products such as antibiotics and physiologically active substances, and because they exhibit various functions in microbial conversion. It has been done. Despite this, there are only a limited number of methods for breeding actinomycetes, and within these limited methods, results have been achieved in improving productivity. . Under these circumstances, it is desirable to establish a DNA recombinant seedling system as a method for research on the breeding and improvement of actinomycetes, and it is hoped that this method will improve productivity and produce new substances. It has become. the current.

Specified bacteria released by mW, @ (S, coelicolor A
1312.

Host-vector systems have been established for S., 1ividlna, etc., and various actinobacterial genes have been cloned. They include, for example, actinorhodin biosynthetic genes (Nature, 309) as genes for the production of antibiotics.
．． 462 (1984)), erythromycin biosynthesis gene (BiOteohnolog7, 2).

808 (1986)). There is a report that a new antibiotic, medelrosin, was produced when the actinorhodin biosynthetic gene was introduced into Streptomyces nis bee, a bacterium that produces the related antibiotic medermycin (A
ntilniOrOl), AgentsChemOth
er, Yu 9, 13 (1986)). In addition, the enzyme gene of actinomycetes is the endoglycosidase H gene (J, Bio
l, 0hecni, 256.10640 (1981)
) There are several reports including

Problems to be Solved by the Invention □ As mentioned above, reports on the cloning of genes of actinobacteria are increasing, but the diversity of abilities of actinobacteria, such as the ability to produce antibiotics and produce 1wL of physiologically active substances, is limited.
-If you think about it, these studies have only just begun.

In particular, genes for enzymes involved in microbial conversion using actinomycetes have not yet been cloned, despite their industrial importance. Therefore, it is desired to use recombinant DNA techniques for actinomycetes to improve breeding of such actinomycetes.

Means for Solving the Problems The present inventors conducted research to provide DNA containing genes related to producers of specific enzymes of Streptomyces used in microbial conversion. the result.

Hydroxylation of the 6β position of ML-236BNa resulted in 08-5'14
We successfully isolated DNA from a strain belonging to the genus Streptomyces that contains a hydroxylase gene that can be converted into . For example, Streptomyces lividans (Streptmyces lividans) lacking or having low hydroxylation activity from ML-236BNa to aS-514.
By introducing a plasmid incorporating the DNA into the vidan.

The gene concerned is expressed, and from ML-236BNa to as-5
Since conversion to 14 is possible, by introducing a recombinant plasmid containing the DNA into Streptomyces carbophilus used for industrial production, for example,
The present inventors discovered that the amplification effect of the gene can be expected to create a strain with high conversion efficiency or a short conversion time per unit substrate (ML-236BNa), and have completed the present invention.

According to the present invention, a recombinant plasmid containing a DNA fragment of the cytochrome P-450 gene, which has a hydroxylase activity that hydroxylates the 6β position of ML-236B Na and converts it to O8-514, is provided. be done.

DNA containing the cytochrome P-450 gene of the present invention@
The pieces were treated with ML-236BNat substrate.

C! As long as it contains a gene for cytochrome P-450 that has a hydroxylase activity that converts it to S-514, it may also hydroxylate other types of substrates. In addition, the origin of the DNA fragment containing the cytochrome p-450 gene is ML-23, regardless of its host.
The chromosomal DNA of actinomycetes, which has the ability to hydroxylate the 61st position of 6BNa to produce O8-514, is preferably used. Such species include, for example, Streptomyces flavovirens (Streptomyces flavovirens).
Examples include flavovirens.

On the other hand, the vector plasmid only needs to be one that can autonomously reproduce within the actinomycete and has excellent stability so that it can be stably inherited by daughter cells when the host cell divides. You can choose freely. Specific vector plasmids include, for example, the known pIff 702 (KatZ at al.,
J, Gen1M1c-robiol, 129.27
03 (1983)).

However, the vector plasmid is the DNA1 of the present invention.
A plasmid having a gene conferring specific antibiotic resistance suitable for screening transformants containing a recombinant plasmid, which can be confirmed as a recombinant plasmid by insertion and inactivation, and which has a wide host range is preferred.

Therefore, such plasmids are designed to express melanin-producing genes even in thiobacteria.
For example, plasmid pMK that can be used in a wide range of hosts.
L16, pMELl 8゜pMEL25 (patent application 1986)
1-193316) is suitable.

The DNA containing the cytochrome P-450 gene provided in the present invention is a vector plasmid containing cytochrome P-450 gene.
Any plasmid that contains DNA containing a gene may be used.For example, a plasmid named by the present inventor and described below may be used.
YO1 or plasmid pHYO2 resulting from recombination of plasmid pHYO1 in host bacteria or plasmid p) derived therefrom! I can mention YO21.

Here, 6β position of ML-236BNa is hydroxylated to form OS-5.
The cytochrome p-450 gene, which has hydroxylase activity involved in the production of ML-14, is ML-2 as shown in the figure below.
36B DNA that is involved in the production of hydroxylase that catalyzes the 6β-hydroxylation reaction of Na.

The recombinant plasmid containing DNA containing the cytochrome P-450 gene of the present invention can be prepared by a method known per se (for example, A, HOpWOO
Genetic manipulation of
streptomyces”, a Laborat
ory Manual, TneJohn Innes
Foundation, 1985 As the vector plasmid, any vector plasmid can be used as long as it can maintain stable replication and growth in actinomycetes, including those derived therefrom depending on the purpose of use.

For example, Streptomyces lividans 5ANK6818
2 [FERMBP 114
1)] using methods known per se (for example, A H
OpWOOd et al., “Genetic Manipul
a-tion of StreptOm7Ces”,A
Laboratory Manual.

The John Engineering Foundation,
Plasmidle J70, which can be collected by (1985)
2 can be used as a vector. In addition, plasmids pMEL1B, pMEL18, and pMEL1B, pMEL18, and pMEL18, which were prepared so that they can be used in actinomycete hosts that cannot express the melanin-producing genes of plasmids pm and T702, are also available.
MEL 25 can also be used in the same way. These plasmids are assigned thiostrepton resistance (hereinafter referred to as TniorJ) and melanin parabiosis (hereinafter referred to as MelJ) as selection markers.

If a transformant exhibiting T1'1iOrl@Mel- is selected, it can be used in the preparation of a recombinant plasmid.

The 6β position of the above ML-236BNa was hydroxylated and O
Chromosomal DNA from a bacterial cell of Actinoma (such as Streptomyces flavovirens) containing a cytochrome P-450 gene with hydroxylase activity that converts to 8-514.
1 Known methods 1 For example, Mar mur's method (J, M
o1. Btol, 3, 208 (19e 1))
Walk away. By cutting the extracted chromosomal DNA 1 with an appropriate restriction enzyme, a DNA fragment containing the desired cytochrome P-'450 gene is obtained together with other DNA fragments. From the mixture of DNA fragments thus obtained, a recombinant plasmid 7 containing DNA containing the desired enzyme
For the i-preparation, the DNAPtr fragment is first incorporated into a vector plasmid that has been treated to be compatible with the defects in the DNA fragment containing the cytochrome P-450 gene. Next, the host bacteria were transformed with the generated six-layer assembly plasmid.

For example, transformants containing a recombinant plasmid exhibiting 'rhior'Mel- are selected. Then, transformants expressing the desired trait are selected from the selected transformants.

An example of using the plasmid pMEL 1g described above as a vector is as follows. That is, chromosomal DNA is digested with restriction enzyme Mbo for 3 to 2
Partially degraded DNA fragments (e.g. '
j', Maniatia et al., “MOleCular
C1on-1ng', Co1a Spring
Harbor Lat) OratOr7゜282 pages, 1
982) and the resulting DNA fragment was
Ifru-circulated pMKL 18 is mixed with pMKL 18, and further ligated with T4 DNA IJ gauze.

This yields a ligation mixture containing the desired recombinant plasmid into which the chromosomal DNA fragment has been introduced. To select the desired recombinant plasmid from the ligation mixture,
The mixture % ML-236BNa was introduced into protoplasts of actinomycetes that do not inherently have the ability to hydroxylate the 6β-position and convert it into O8-514, or have only an extremely low lateral force.

Spread on an agar plate. Next, a soft agar medium containing thiopeptin is overlaid on the cultured agar plate. After overlaying, the agar plate is cultured, and a transformed strain exhibiting 'rh i Or' grows. Among these, the transformed strain containing the recombinant plasmid does not produce melanin and can be easily identified.

Next, the selected transformed strains that do not produce melanin are M
The colonies are transplanted onto an agar plate supplemented with L-236B Na and cultured to allow sufficient colony growth.

Agar plugs are punched out using this colony wotrocker. The 10 agar plugs prepared in this way were put into a microtube as one group, and the ethanol aqueous solution was
After stirring well and extracting, the supernatant was subjected to high performance liquid chromatography (hereinafter referred to as l'-HPL (!J
Attached to that. Next, the sample was subjected to a primary screening to determine the presence or absence of a peak derived from C9-514.

Two-year screening is one-doctor screening C8-51
Colonies included in the population in which the presence of a peak derived from 4 was observed were inoculated into a medium containing thiopeptin and cultured with shaking. Next, after adding M and L-238B Na and continuing the shaking culture, the culture solution is collected into a microtube, centrifuged, and the supernatant is collected. Collected supernatant yB Hp:c, ML-236BNa 6
Clones in which the β position is hydroxylated and converted to C8-514 are selected. In this way, a clone producing 14% of C8-5 is the cytochrome P-4 having hydroxylase activity of the present invention.
A recombinant plasmid containing DNA containing 50 genes is maintained. Therefore, if this is extracted using the plasmid extraction method described above, the vector plasmid will contain ML-236BN.
D containing a cytochrome P-450 enzyme having hydroxylase activity that hydroxylates the 6β-position of a and converts it to O8-514.
A recombinant plasmid containing NA can be obtained. The recombinant plasmids thus obtained include, for example, plasmid pHYO1 or plasmid pH
YO27i: Specific description of 31 recombinant plasmids Recombinant plasmid pHY obtained by the above method and Plasmid I) HYO2 (see Examples and Figures 2 and 3), and further derived from plasmid pHYO2. Plasmid pHYO 21 (
(See Figure 4) will be described in detail.

(1) Confirmation of hydroxylase activity by plasmid pHYO1 The inserted DNA fragment contained in plasmid pHYO1 hydroxylates the 6β position of ML-236BNa and converts it into O8-51.
Cytochrome P- with hydroxylase activity converting to 4
The inclusion of 450 enyunzi can be understood from the following. That is, it is understood that the plasmid removal method, which occurs by removing this plasmid from a recombinant strain containing the plasmid pHYOf, results in sensitivity to thiopebutin, which is accompanied by the simultaneous loss of hydroxylation activity and the loss of the absorption maximum near 4SQ nm in the co M spectrum. Ru. Furthermore, when the plasmid removal method is used for retransformation using plasmid pHYO1 as a host, all retransformed strains obtained are Th
This was confirmed by the fact that the hydroxylation activity became 1 or, the return of the hydroxylation activity and the return of absorption 1 near 4 50 nff1 by the CO difference spectrum, and that plasmid pHYO1 could be isolated from this retransformed strain. However, as is clear from FIG. 2, this plasmid pHYO1.

This is inconvenient for miniaturization. Plasmid pHYO2 described below was used for miniaturization research (n) Confirmation of the presence of plasmid pHYO2 and plasmid pHYO21
ML-2
Among the recombinant plasmids isolated from one of the transformants exhibiting the ability to convert to 36B Na'i'C8-514, it was found that in addition to plasmid pHYO1, there was also a plasmid pHYO2 of approximately the same size. That is,
Plasmid pHYO1 was purified by Sac I digestion to approximately 10
The plasmid mixture is known to produce DNA fragments of about 10 kb and about 5.8 kb derived from plasmid pHYO1 by digestion (see Figure 2). In addition, approximately 13.1 kl derived from plasmid pHYO2)
and generates a DNA fragment of approximately 2.4 kb. Therefore, the plasmid mixture was used to transform Streptomyces fistulae, and by Sac I digestion, approximately 13.1
A transformant strain containing only plasmid pHYO2 can be obtained by isolating a transformant strain containing only plasmids producing DNA fragments of 24 kl) and 24 kl). This transformed strain was developed by ML-2s 6 B Na71) et al.
Because it has the ability to convert to S-514, plasmid pHYO2 also converts to ML-S-514, similar to plasmid pHYO1.
It has an inserted DNA fragment containing a cytochrome P-450 enzyme having hydroxylase activity that hydroxylates the 6β-position of 236BNa and converts it to C6-514. Plasmid pHYO2 can be prepared from a transformed strain containing the plasmid alone.

By creating a restriction enzyme cleavage map of the plasmid pHYO2 shown in Figure 3, it was found that approximately 3001) 1) containing the Sph I site present in the vector part of the plasmid is deleted, and that it has hydroxylase activity. In the inserted DNA fragment of about 10 kl) containing cytochrome P-450 genes, about L1 containing at least about 6.7 kl) from the sac I to the Sph 1 site in the host box.
It is understood that the DNA fragment of kb underwent recombination and was ligated in the homologous region or in the opposite direction to the relevant portion in the inserted DNA fragment of pHYOl. In this way, at least about 6.7 kl) of DNA [ML-2363N] is C! Undergoing conversion to S-514 converts ML-236BNa to O8-5
A cytochrome P-450 gene with hydroxylase activity converting to 14 or at least Sac, 1 to Sp
Approximately 7.1kk including approx. 6.7kl to h l site)
) and that it also contains the promoter region of the cytochrome P-450 gene.

In plasmid pHYO2 as described above.

Approximately 6.7 kb from Sac l to Sph l site
This means that the approximately 2.4 kb DNA fragment generated by SacI digestion contains a cytochrome P-450 fragment with hydroxylase activity in the DNA fragment of approximately 7.1 kt) containing hydroxylase activity. This indicates that it is not necessary for the expression of oxidase activity. Therefore, this approximately 14 kl) D
It is readily understood that plasmids can be miniaturized by removing the NA fragment. First, plasmid p
HYO2 was digested with Baal and it was about 13.1 kb and about 1
4kl) of DNA fragments were obtained and then heat-treated.
Ligate with 4 DNA IJ gauze to obtain a ligation mixture. Next, it is introduced into Streptomyces lividans protoplasts, and a transformed strain is obtained by the method described above. Furthermore, these transformed strains were cultured by the same method as in the secondary screening described above, and clones producing C8-514,i were selected by HPLC. Grasmids can then be extracted and obtained from the selected clones by the plasmid extraction method described above. The specific product thus obtained is shown in Figure 4, consisting of about 13.1'kb, Sac
Plasmid pHYO21 with one I cleavage site
・Figure 4 shows Grasmid pHYO
A restriction enzyme cleavage map of 21 is shown, which shows that the plasmid is identical to plasmid pHYO2, except that a region corresponding to 1 to 2.4 kb DNA fragment generated by SaCl digestion in Grasmid pHYO2 has been removed. .

(IOI Determination of cytochrome P-450 gene localization region Grasmid p) Insert DNA fragment of rYO21 approx.
As already mentioned, cytochrome P-450 having hydroxylase activity that converts ML-2368Nm to CS-514 is located within 1 kb. Next, in which region within this approximately 7.1 kb inserted DNA fragment is Cytrome P inserted?
The localization of the -450 gene was examined. The investigation method was carried out using restriction enzyme sites within the inserted DNA fragment. That is, p) After completely digesting rYo 21 with Sph t, when a is subjected to low-glue electrophoresis, approximately 11
．． It can be seen that the DNA fragments were cleaved into DNA fragments of 6 kb and approximately 1.5 kb.

The DNA fragment containing this approximately 11.6 kb DNA fragment was excised, the DNA fragment was obtained by electroelution method, and this DNA fragment was injected into T4D.
After ligation treatment with NA ligase, it is introduced into Streptomyces lividans protoplasts.

The thus obtained transformed strain contains plasmid pHYO21.
Approximately 1.5 kb of 5p from the approximately 7.1 kb inserted DNA fragment of
It contains an approximately 11.6 kb plasmid pHY022 containing an approximately 5.6 kb inserted DNA fragment in which the ht fragment has been deleted.

In addition, when a sample of plasmid pHYO21 completely digested with Mlul was subjected to glucose gel electrophoresis, it became approximately 8.6 kb.
, approximately 3.6 kb (approximately 0.3 kb of vector DNA fragment)
b) and was cleaved into a DNA fragment of approximately 0.9 kb. Approximately 8.6kb of DNA in this
The DNA fragment containing the fragment was cut out and the DNA was analyzed by electrophoresis.
I got the fragment, this is '1T4DNA! After ligating with j gauze, the mixture is introduced into Streptomyces lividans protoplasts. The thus obtained transformed strain is pHYO2
The plasmid pHYO 23 contains an approximately 8.6 kb plasmid pHYO 23 containing an approximately 2.9 kb insert DNA fragment obtained by deleting the approximately 4.2 kb DNA fragment generated by MluII digestion from the approximately 7.1 kb insert DNA fragment of 1. There is.

Next, the transformed strains containing these plasmids pHYO 22 and plasmid pHYO 23 were cultured by the same method as the secondary screening described above, and HP
At the same time, the amount of C8-514 produced was examined by LC, and the cell-free extract obtained by ultrasonic disruption of bacterial cells and centrifugation was analyzed using the method of Ohmura et al. (J, Biol.

Chem., 239, 2370, 1964).

The presence or absence of cytochrome P-450 was determined by differential Nuvector.

FIG. 7 shows the results, and it is shown that the cytochrome P-450 gene is localized within the approximately 2.9 kb inserted DNA fragment of pHYO 23. Also, ML-236B N
approximately 7°1 kb to provide the host Streptomyces lividans with sufficient conversion activity from a to C8-514.
It is shown that the entire range of is required.

The detailed description of the actinomycetes used in the present invention is as follows.

1. Streptomyces flavovirens 5ANK 6
3684 ML-236B used in the present invention Nm to C8
The morphological properties and physiological properties of Streptomyces flavovirens 5ANK 63684, which has the ability to convert into -514, are as follows. In addition, preparation of various agar media,
Seed culture, main culture, and observation of results were conducted in accordance with ISP standards, Applied Microbial Industry Examination Standards, and Waxman's recommendations.

The color tone of growth on various media was in accordance with the "Color Standards" (Japan Color Research New Edition).

l) Morphological characteristics Morphological characteristics were observed not only by optical microscopy but also by electron microscopy using samples prepared in the manner described below.

For fixation of bacterial strains, 2% osmic acid was used to fix the strain at about 1O at room temperature.
Time steam fixed. Leave the fixed sample on the agar medium, 5
Dehydrated with ethanol at each concentration of 0,70,80,90.95°100% for 15 minutes, and infiltrated with rI in amyl acetate medium.
I changed it to t. For drying, critical point drying was performed using a critical point drying device HCP-1 using liquid carbonic acid as a transition liquid.

The dried sample was deposited using an ion coater fB-3 so that the gold film had a thickness of about 200X. The observation was carried out using a scanning electron top microscope MSM-4 model (Hitachi Akashi). The acceleration voltage at this time is 25 kV.

Table 1 Morphological characteristics (28°C, 10 days observation) 7-・' 5, / /' 2. Culture characteristics on various media Table 2 (28°C, 14th day observation) G: Growth AM: Aerial mycelium R: Back side SP: Soluble dye 3. Physiological property table 3 Starch hydrolysis Positive 1 Medium 1:
Tryptone Yeast Extract Broth (ISPI) 2 Knee 27D Tons Yeast Extract Iron Agar (ISP 6)
3: Terocin agar (ISP7) 4: Yeast malt extract agar (ISP2) 4,
Carbon source assimilation property table 4 廿: Well assimilated +: Assimilated - Not assimilated 5,
Cell wall chemical composition using the method of Ecker et al. (Appl, Microb
Iol, 12, 236*1965), LL-diaminopimelic acid and glycine were detected as major constituents of the cell wall. The cell wall type is type I.

To summarize the above results, 5ANK 63684 shows a straight to curved sporophyte, and a chain of 50 or more spores is formed at the tip. The spore surface is smooth. Substrate mycelia grow pale olive to olive yellow to light brownish gray, and epiphyte grayish white to olive gray to gray aerial mycelia. Aerial hyphae are powdery and black spots may be seen in the later stages of cultivation due to hygroscopic growth. Also, soluble pigments and melanin-like pigments are not produced. Cell wall type is LL
-Contains DAP and glycine! It is a type.

From these properties, it is clear that S-63684 belongs to the genus Streptomyces. Among the known Streptomyces genus, Streptomyces 7 is a particularly near-green species.
Lapovirence is mentioned. Therefore, a simultaneous comparative culture of Streptomyces flavovirens strain ISP 5062 and this strain was conducted. As a result, no differences were observed between the two strains in terms of morphological and physiological properties. Therefore, 5ANK 63684 is considered to be the same species as Streptomyces flavopyrene, and this strain was referred to as Streptomyces flavovirens 5ANK6368.
It was identified as 4.

2. Streptomyces lividans 5ANK 6818
2 [FERM BP-114
1) ) Streptomyces lividans 3131 carrying plasmid pIJ7Q2 constructed by E. Katz (J. Gen. Microbiol.
-129, 2703-271 (+1983).

It is Streptomyces lividans TK 21.

This strain is “Genetic Manipulation”
n of Streptomyces#*A Labo
ratory Manual, The John I
Nnea Foundation+1985 K, and is used worldwide as a host for actinomycetes.

Plasmid pMEL1 constructed by the inventors
8 (JP-A-62-122585 #J, Antib
ioticg.

◎BP-, which is a Streptomyces lividans TK21 strain that carries
1140) ] The morphological and physiological properties of this strain are described in detail.

Streptomyces flavovirens 5ANK63684
(Feikoken Bibori No. 9170) was mixed with GGCy liquid medium (0,
4 tiglycerol, 0.1% glycine, 0.4% cademino acid, 0.1% magnesium sulfate, 0.01% calcium chloride, 0.1% yeast extract, trace metal salt solution 4 mVl
) and cultured with shaking at 28°C for 3 days. This was used as a seed, and a 5% amount of the seed was inoculated into a fresh Lasco and cultured at 28°C for 24 hours on a reciprocating shaker. Mycelia were obtained from this culture solution by low-speed centrifugation, and the method of Marmur (J.

Mol・B101・、Yu・208. (1961)), total DNA was extracted and purified using DNA lysis buffer (1961).
10mM Tri day (pH 7,5), 10 InM
The DNA was dialyzed against NaCl (1mM EDTA) to obtain donor DNA.

5 μg of the donor DNA obtained in this way was
of Mbo I at 37°C.

It was partially digested into DNA fragments of 3 to 20 kb. This reaction solution was treated at 70° C. for 10 minutes to deactivate Mbol by heating. Next, 120 times the capacity
After adding ethanol at -70℃ for 30 minutes,
The DNA was precipitated by centrifugation at 5,000 rpm for 3 minutes.

On the other hand, bester plasmid pMEL18 (see reference side).

This plasmid is Streptomyces lividans 5ANK.
60587 (Feikoken Bibori No. 9168) Karari, A.
Hopwood et al.”Genetic Manipula
tion of streptomyces'', A L
abo14tory Manual, TheJohn
Innes Foundation (1985). ) 1μm of 5 8g
The mixture was reacted for 2 hours at 37° C. for complete cleavage.

This Bgl II-cleaved pMEL 18 was prepared at 70°C.
After heating for 10 minutes to inactivate Bgl.

Ethanol precipitation was performed in the same manner as above.

Donor DNA partially digested with Mbo prepared as above and pMKL18 completely digested with Bgl I [
was dissolved in 35 μl of distilled water. Add to this a 10x gauze reaction buffer (8601nM Tris-HC).
I, 6g1■M MgCl2. pH7,6)5fil
, 5 μl of 50 mM dithiothritol and 10
Add 5 μl of ff1M ATP to bring the total i1 to 50 μl, add 6 u of 'r4 DNA ligase, and add 14 μl of ATP.
The reaction was carried out at ℃ for 16 hours. In this way pMKL 1
8 and Streptomyces flavovirens 5ANK636
A recombinant plasmid mixture of 84 chromosomal DNA was prepared.

To separate the desired 7 recombinant plasmids 7i:A from this recombinant plasmid mixture, ML-236BNa%
Streptomyces lividans 5ANK63086 is an actinomycete that does not have the ability to convert to O8-514 or has only a very low ability to convert it to O8-514.
The recombinant plasmid mixture was introduced into protoplasts (No. 169).

That is, 3.5 g at 28°C in GGC7 medium containing 34% g sugar.
Streptomyces lividans 5ANK6 cultured for days
Seed culture medium containing 3088 mycelia, 34% fresh
A 5% amount of the cells was inoculated into 100 ml of sucrose-containing GGCy medium (500 ml Sakaro flask), and cultured with reciprocating shaking at 28° C. for 24 hours. A mycelium pellet was obtained from the culture solution by low speed centrifugation.

This>7;; 20 at P medium (320 mg sugar,
25mM TKS buffer ih70mM NaCl, 1
0 ff1M MgCl2.6H20゜20mM C
aCl2.2H20) and washed. Then, the mycelium obtained by centrifugation was suspended in 20 tul of P medium. To this mycelial suspension, 1 ffie of a 40 my/rttlaK lysozyme solution was added, and the eggs were incubated at 28° C. for 1 hour to generate protoplasts of Streptomyces lividans 5ANK630B6 strain. This mixture of protoplasts and undissolved mycelia was filtered naturally through a glass filter (3G3) to obtain a protoplast solution containing a large amount of protoplasts. This was centrifuged at low speed and the pellet was resuspended in P medium. This operation was repeated three times and thoroughly washed to prepare a protoplast solution containing the desired number of protoplasts.

The obtained protoplast solution was centrifuged to obtain a pellet.

The previously obtained recombinant plasmid mixture was added to this and gently stirred to uniformly disperse the protoplasts. P medium 05 containing 20% polyethylene glycol 1540% was added to this and left to stand for 1 minute.
of P medium was added. All transformation operations were performed at 0°C. After transformation, protoplast pellets were obtained by centrifugation. Add 5 rug of P medium to this and stir thoroughly.
Washed and centrifuged. This operation was performed at least three times, and the transformed protoplasts were suspended in the desired amount of P medium and spread on a regeneration medium (R2MP agar plate).

R2MP medium (sucrose 12 Of, K2S0a 0
125f.

K2HPO40,05f, MgR2#6 R20
10,12f, CaC1z"2H202,95f
, Glue: I-su 4f, casamino acid 0.1, L-proline 3f, DL-norleucine 0.05f, tyrosine 0.
5F, Yeast extract 2F, Malt extract 5J, 250m
MTES buffer (pH 7,2) 10'0rttl, trace metal salt solution@2at, agar 20f7z added 100
0I111) is a medium prepared to emphasize the production of melanin-like pigments. After spreading on an R2MP agar plate, the plate was incubated at 28°C for 20 hours. Soft agar R5 medium (120% sucrose, K2HPO) to which thiopeptin was added to a final concentration of 50 μf/me
40,2f, MgR2”6 R20 & 1f,
CaCl2 ・2H2012f, 250mMTl!
S buffer (pH 7,2) 100 ml, glucose: l-su 10 f, #mother extract 4 f, polypeptone af, K
clo, 5f.

3 tugs of agar (add 5 f agar to make 1 oooag) were layered. After overlaying, the agar plate was cultured at 28°C for 7i. Since the strain that does not produce melanin (Mel- strain) is a transformed strain that has a recombinant plasmid, &Mel
- Strains were selected.

-The next screening was carried out as follows. That is, the selected transformants exhibiting 1Me knee were added to a final concentration of 300 μl/
Transferred onto GPY agar plates (2 Tiglucone, 1 Tivolipeptone, 0.1% yeast extract, 2 Ti agar, pH 7,0) supplemented with ML-23aBNaz and 2B' (with 7.0%).
The colony was cultured for ~10 days to allow sufficient growth. These colonies were punched out with a Trotzker to produce agar plugs with a diameter of 1.5 mm. Ten agar plugs prepared by punching out each colony in this way were considered as one group and 1.
Pour into a 5 ml microtube and add 200 μl of 20% ethanol! Added 1 round, stirred well, and extracted.

Next, the supernatant was centrifuged at 15.00 rpm for 5 minutes and subjected to HPLC, and the presence or absence of a peak corresponding to O8-514 was determined for primary screening. HP
LC was carried out using Radial Pack CI8 as a column and a mixed solvent of 25% acetonyl (IJ) and 0.1% triethylamine (pH 3.2; prepared with phosphoric acid) as a mobile phase at a flow rate of 2 ml/min.

The secondary screening is O8-5 in the -second screening.
Thiopeptin 25μf/10 colonies included in the population in which the presence of a white rice peak was observed in
me'f-containing GPY liquid medium and incubated at 28°C for 3
The cells were cultured with shaking for days.

Next, ML-236BNa was added to a concentration of 300 μf/ml, and the shaking culture was continued at 28° C. for 2 to 3 days. The culture solution was collected into a 1.5 ntl microtube, centrifuged at 15.00 rpm for 5 minutes, the supernatant was collected and subjected to HP and LC, and the 6β position of ML-236BNa was hydroxylated and converted to OS-514. We selected clones that had The HPLC conditions were the same as the primary screening, but depending on the situation, the mobile phase was a mixed solvent of 30% acetonitrile and 0.1% triethylamine (pH 3,2; prepared with phosphoric acid), and the flow rate was changed to 1 ml/min. I did it.

By the screening method described in Transformation Example 2, M
The 6β position of L-236BNa is hydroxylated.

Transformant strain Streptomyces lividans MLR-1528No, which is endowed with the ability to transform into O8-514,
416 stocks were selected. This is because this strain contains a specific plasmid (plasmid pHYO1).
It is considered that it has become possible to convert to L-236BNa7iC8-514. This plasmid pHYO1i
It was prepared and confirmed by re-transforming Streptomyces lividans.

That is, 20 ml of GGC7 medium containing 34% sucrose
z 50 rxl in a flask with a branch, and add ML to it.
After inoculation with R-1528No, 416 strain mycelium.

Cultures were incubated at 24-28° C. for approximately 72 hours on a reciprocating shaker at 120 rpm. It then entered a 500at Yosaka flask. 1 o G G Cy medium containing 34% sucrose
A suspension of the above seed culture was inoculated in an amount equivalent to 1 to 5% of the medium, and cultured at 24 to 28° C. for 24 to 48 hours with reciprocal shaking.

This culture solution was centrifuged at low speed (for example, 10,000 f).

The mycelium was collected at 4°C for 20 minutes, and the supernatant liquid was removed with a slant to obtain a mycelium pellet. The mycelial pellet was dissolved in 20 m/s TES buffer (25 mM), 25 mM I!DTA and 25
lysozyme solution at a concentration of 40 mq/me was added to this resuspension.
ml and warm this mixture slowly (while stirring) at 37°C for 5 to 15 minutes.

Next, add 3 ml of 10% sodium dodecyl sulfate (
SDS) solution was added, mixed gently, and then heated at 37° C. for 5 minutes to lyse the bacteria.

This lysate was then centrifuged at 40,000'i, 4°C, for 30 minutes to obtain a crude lysate as a supernatant.

Add 1/4 volume of 5M salt to this to bring the final salt concentration to 1.
'M, and when cooled at 0℃ for 2 to 3 hours, the previously added SD
3000S' as S precipitates.

Centrifugation was performed at 0°C for 15 minutes to remove SDS.

Ribonuclease was added to this supernatant and digestion was carried out at 37°C for 20 minutes. Pronase was then added and digestion was carried out at 37°C for 20 minutes. Add 40 polyethylene glycol (PKG) to this digestive fluid.
) 6000 solution to a final concentration of 10%,
If this mixture is kept at 0°C overnight, some DNA will precipitate, so after gentle centrifugation (3000f, 0°C, 15 minutes), discard the supernatant.

The precipitate was suspended in 4.7 ml of TES buffer, thoroughly dissolved, and dialyzed in the TES buffer to obtain a DNA extraction sample.

Cesium chloride was mixed with the DNA extraction sample obtained in this way, and the fluorescent coloring agent ethidium bromide (F:
TBr ) i was added and mixed to prepare a solution with a density of 1.620. This solution is 150,000f.

Equilibrium gradient centrifugation was performed at 18° C. for 40 hours.

When this centrifuge tube is irradiated with 320 nm ultraviolet light.

In the centrifuge tube, it was found that the DNA of the closed circular plasmid pHYO1 was separated as a fluorescent band under the strong fluorescent band of the linear DNA derived from the chromosome.

Collect the fluorescent band part of the closed circular plasmid DNA, extract it three times with an equal volume of n-butyl alcohol to remove ethidium bromide, and then add the aqueous layer to an appropriate buffer (e.g. +10ff1M) IJ. vinegar.

tomM NaCl and 1 mM EDTA, pH=7
．． 5) to dialyze the pure recombinant plasmid pHYO.
I got 1.

The pure recombinant plasmid pH thus obtained
YO 1 is ultraviolet! The concentration was determined from the absorbance at 26 onm.

The size of the inserted DNA fragment in the recombinant plasmid pHYO1 was calculated by agarose gel electrophoresis. That is, limit one recombinant plasmid to 0.5 μm #cumulativeB
By cutting with cl l.

D inserted into vector plasmid pMKL 18
The size of the NA fragment can be determined. The size of the inserted DNA fragment was found to be approximately 10 kb.

Lambda DNA Hincl [
The sizes of the five DNA fragments generated by Bcl I digestion were measured from the electrophoretic mobility using the i-cut fragment or the Haq m-cut fragment of φx174 DNA. The total of these (approximately 15 Jkb) and vector plasmid pMEL
Insert the difference between the size of 18 (approx. 5.1!1cl))
It was the size of A fragment.

In addition, the recombinant plasmid pH obtained in this way
Streptomyces lividans 5ANK63086 by the method described in Example 1 using YO1 (see Figure 2).
(Kaiko Kenzonoyori No. 9169) was re-transformed. When the ability of converting ML-236BNa to C8-514 was examined for the 50 transformants obtained by this re-transformation, all of the strains The conversion ability was confirmed.The plasmids isolated from these viruses were pHYO.
It was l.

Removal of plasmid pHYO1 from and transformation with pHYOl Plasmid p) fYOl was introduced into ML by
Strain 416F-7, in which plasmid pHYO1 was shed and removed by acriflavin or protoplast regeneration, was obtained from transformant strains MLR-1528No and 416, which were endowed with the ability to convert -236BNa to 08-514. This strain was sensitive to thiopeptin with removal of plasmid pHYO1. Furthermore, when this strain was examined for its ability to convert to O8-514 using the same method as the secondary screening described in Example 2, no conversion activity was observed. Furthermore, when the CO difference spectrum of this strain was measured using the cell-free extract mentioned above, the maximum absorption near 450 nm had disappeared. On the other hand, in the control method, MLR-
In 1528No. and 416 strains, conversion activity and C
A maximum absorption near 450 nffl was observed in the O difference spectrum.

This plasmid deleted strain 416F-7% was then transformed by the method described in Example 1 using plasmid pHY017i-. The transformed strain obtained by this transformation is resistant to thiopeptin.

Furthermore, when the conversion activity to O8-514 was examined by the same method as the secondary screening described in Example 2, the conversion activity was restored. Furthermore, when the CO difference spectrum was measured, the maximum absorption near 45 Onffl had also returned. This indicates that the converting enzyme that hydroxylates the 6β-position of ML-236BNa and converts it to O8-514, that is, the hydroxylase, may be cytochrome P-450, and that the progeny of this cytochrome P-450 gene is a plasmid. pHYO
This shows that it is present in the inserted DNA fragment of No. 1.

As described in Example 3, it was confirmed that all of the 50 transformed strains obtained by re-transformation with plasmid pHYO1 contained plasmid pHYO+, but one of these strains had plasmid pHYO+. pHYO1'i
Analysis of DNA fragments produced by Sac I digestion revealed that this is a mixture of plasmids containing two types of plasmids of approximately the same size.

That is, pHYOI is reduced to approximately 10k by Sac I digestion.
l) and approximately 5.8 kt), but
The mixture was transformed into plasmid pHYO1 by SaCl digestion.
In addition to approximately 10 kl) and approximately 5.8 kl) DNA fragments derived from
3.1 kl) and a DNA fragment of approximately 14 kb was generated. Therefore, in order to isolate the plasmid pHYO27z, transformation was performed using the mixture according to the method described in Example 1, and a plasmid was prepared from the grown transformant according to the method described in Example 3. Then the plasmid 7
< Approximately 13.1kt by digestion with Sacl
) and approximately 14 kbcr) were selected, and a transformant containing only plasmid pHYO2 was obtained. The ability of the transformed strain containing the plasmid pHYO2 alone to transform MI, -236BNa to O8-514 was determined by the secondary screening method described in Example 2, and it was confirmed that it retained the transforming ability. confirmed.

Example 6. Preparation of plasmid pHYO21 Plasmid pHYO2 contains about 1 cytochrome p-450 gene with hydroxylase activity, according to its restriction enzyme cutout map.
In the case of a 0 kb inserted DNA fragment, the distance from at least SaCl to the sph l site is about 6.7 in the host bacterial cell.
A DNA fragment of approximately 7.1 kb containing plasmid pHYO 1 was recombined, and the homologous region to the plasmid pHYO 1 was oriented in the opposite direction (see Figure 3). At least Elac
Approximately 7 including approximately 6.7kb from l to Sph l site
．． The fact that ML-236BNa underwent conversion to C9-514 even though the 'b DNA fragment was coordinated to 1 in the reverse direction suggests that the water that am Cytochrome P-45 with oxidase activity
This is a miniaturized plasmid pHY from which the approximately 7.1 kb DNA fragment is removed.

:L'! The preparation was carried out as follows.

Plasmid pHYO2 was digested with lμf% 5u of 5acl for 2 hours at 37°C. Next, the mixture was heated at 70° C. for 10 minutes to inactivate SaCl, and then ethanol precipitation was performed. Next, this SaCl-digested ethanol precipitate was dried and dissolved in 35 μl of distilled water, and 5 μl of 10x ligase reaction buffer, 5 μl of 50 mM dithiothritol, and 51 tl of 10 mM ATP were added to bring the total volume to 50 ttll. And so. Add 2 u of T4 DNA ligase to this.

The reaction was carried out at 14°C for 16 hours. In this way, the Sacl-digested DNAvgr fragment of plasmid pHYO2 was self-ligated, and this was introduced into the protoplasts of the plasmid pHYO1 removed (strain 416F-7) described in Example 3 by the method described in Example 1. Shis T
A transformed strain exhibiting h1or was obtained. Plasmids were then extracted from these transformed strains by the method described in Example 3, and a plasmid with seven approximately 2.4 kb DNA fragments missing, ie, a plasmid with a size of approximately 13.1 kb, was selected. The plasmid thus obtained is S
Plasmid PHY of approximately 13.1 kb with one acl cleavage site.

21 (see Figure 4). This plasmid is pHYO
Similar to ML-236B 2, it contains a cytochrome P-450 gene that has a hydroxylase activity that hydroxylates the 6β-position of Na and converts it to C8-514.

At least about 6 from Sac l to Sph l site
．． It has a DNA fragment of approximately 7.1 kb, including a 7 kb portion.

Like plasmids pHYO1 and p)tYO2, they confer on the host cell the ability to convert ML-236BNa to C8-514.

Example 7. Preparation of plasmid pHYO22 (plasmid)
'pHYO2105μ, 9 f 15u+7) Sph
Digestion was carried out using I2 at 37°C for 2 hours. Then, after heating to 70°C for 10 minutes to inactivate sph l, 0.84
7. It was subjected to Kers-Glue electrophoresis. D of about 11.6kb and about 1.5kb in agarose gel electrophoresis.
NA fragments were generated, from which a group containing a DNA fragment of approximately 11.6 kb was excised, and the DNA fragment was eluted by electroelution. Add 5 μl of 10x gauze reaction buffer to 35 μl of eluate, and 5 μl of 50 mM dithiothritol! , and 5fil of 10 mM ATP to bring the total volume to 50 μl, and add T4 DNA ligase 2 to this.
ut, and reacted at 14°C for 16 hours. In this way, the approximately 11.6 kh DNA fragment generated by Sph 1 digestion of Grasmid pHYO21 was self-synthesized, and this was transferred into the protoplasts of the 416P-7 strain described in Example 3 as described in Example 1. A transformed strain exhibiting Th1o' was obtained. Then, a plasmid was extracted from this transformed strain by the method described in Example 3. The plasmid obtained in this way is S
It is a plasmid PHYO22 (see Fig. 5°) of approximately 11.6 kb that has one ph I cleavage site.

This plasmid has an insert DNA fragment of approximately 5.6 kb, which is obtained by removing approximately 1.5 kb generated by sph I digestion from the approximately 7.1 kb insert DNA fragment of pHYO21. ML-2 to
36B cannot provide the ability to convert Na to C8-514.

Example 8. Preparation of plasmid pHYO23
'pHY02105μ, 9T! Digested with 15u (DMlu I) at 37°C for 2 hours. Then, after heating to 70°C for 10 minutes to inactivate Mlu I, it was subjected to 0.84 agarose gel electrophoresis. It is about 8.6 kb, about 3.6 kb (vector
containing approximately 0.3 kb of DNA fragment) and approximately 0.9 kb
A DNA fragment of approximately 8.6 kb was generated, and a group containing a DNA fragment of about 8.6 kb was excised, and the DNA fragment was eluted by electroelution. Add 35 μl of this eluate and 5 μl of 10x concentrated reaction buffer! ! , 5μg of 50mM dithiothritol and 5μg of 10mM ATP were added to bring the total volume to 50μl.
was added and reacted at 14°C for 16 hours. In this way, the approximately 8.6 kb DNA fragment generated by MluI digestion of plasmid pHYO21 was self-cyclized,
This was introduced into protoplasts of the 416P-7 strain described in Example 3 by the method described in Example 1.
A transformed strain showing io' was obtained. Next, a plasmid was extracted from this transformed strain by the method described in Example 3. The plasmid obtained as follows is M
Plasmid 1 of approximately 8.6 kb with one lu 1 site
) HYO23 (see Figure 6).

This plasmid is produced by Mlu I digestion from the approximately 7.1 kb inserted DNA fragment of pHYO21.
．． The host cell (416P-7 strain) K ML-2 has an insert DNA fragment of approximately 2.9 kb from which approximately 3.6 kb including 9 kb and the vector DNA fragment of approximately 0.3 kb has been removed.
36B Provides the ability to convert Na to C8-514. However, its ability is about 1/3 compared to the ability imparted by pHYO21.

Test Example 1 Imparting the ability to convert the following strains (a) Streptomyces lividans 5ANK63
(1) Streptomyces lividans 5ANK 63086 containing the vector plasmid pMEL 18i;
(C) Streptomyces lividans 5ANK63
G86 strain ML-236BNa according to the present invention was transformed into CB-5
Cytochrome P with hydroxylase activity converting to 14
- MLR-152, a strain transformed with the recombinant plasmid pHYO1 containing 450% DNA
8. NO416 strain; ((L) MLR-1528-NO416 taste to plasmid p) 4111F-, a strain from which iYo 1 was removed
7 strains; (e) Plasmid pHYO of 416F-7 strain
RTF-85 strain, which is a retransformed strain by 11c; (f) Blasmi l' pHYO2 of 416P-y strain;
Strain RTF-182, which is a retransformed strain of 1ic; Streptomyces lividans 5AN in Zarauchi (a)
K63086 strain and (d) 416P-7 strain were inoculated into GPY medium, and (b) Streptomyces lividans 5A
NK60587 strain, (C) MLR-1528N0.4
18 strains, (e) RTF'-85 strain and (f) RT
Strain F'-182 contains C containing 25 μf/at of thiopeptin.
) Inoculated in PY medium.

Shaking culture was carried out at 28°C for 3 days. Next, this was used as a seed to inoculate fresh GPY medium in an amount of 5%. Then 2g
'l: ML-236BNa was added to the culture solution after shaking culture for 1 day.
Added to 500 μf/ffi/@ degree and further added 2
Shaking culture was carried out at 8°C for 3 days. Next, collect each culture solution into a 1.5-capacity microtube. 15. Go.

After centrifugation at rpm for 5 minutes, the supernatant was collected and subjected to HPLC
C8-514 produced by was measured.

The production amount of C8-514 is (a) 1 pf/at, (b
) is 5μ2/IItl & (C) is 359μf/d, (d
) is 1af/ml, (e) is 340 ytlat
, (f) was 390μf 711g. This is true for plasmid pHYO1 and plasmid pHYO2.
1 or originally hydroxylated at the 6β position of ML-236BNa to form CS-
ML-236B does not have the ability to convert to Streptomyces lividans 5ANK63086, a host with extremely low ability.
) This indicates that the ability to convert Ja to O8-514 is imparted.

Transformation of Streptomyces lividans with plasmid pHYO1 and plasmid pHYO21 5ANK
63086 and Streptomyces lihydans 5AN
[ML-236BNaiC8-514 such as K6G587
The method for preparing cell-free extracts for ``determination of cumulative activity'' and ``measurement of cytochrome P-450'' was carried out as follows.

The strain precultured in GPY medium was transferred to fresh GPY medium.
Ox/ (in a 50 oat Erlenmeyer flask) was inoculated in a 5% amount and cultured with rotational shaking at 28°C for 24 hours.

ML-236BNa was added at a concentration of 500 μf/ml, and the rotary shaking culture was continued at 28° C. for 24 hours.

5. Incubate the culture solution at 0°C. Bacteria were collected by centrifugation in QOOXf for 15 minutes. After washing three times with 0.85% saline cooled in water, 80 m
M) Lis-hydrochloric acid buffer (pH 7.4, hereinafter referred to as "A buffer")
) is added.

The water-cooled lower layer was sonicated. Then 2G, QOOXf is 30
After centrifugation for a minute, the supernatant was collected and used as a cell-free extract.

The method for measuring hydroxylase activity from ML-236BNa to t-514 was performed using 6 volumes of cell-free extract under the following conditions (reaction a composition): Cell-free extract 0.8 m/NADPH regeneration system NADP 0.26 a/ Glucose 6-phosphate 14mM glucose 116-
Phosphate dehydrogenase W0.5u Nicotinic acid amide
10mM magnesium chloride z
smM Ferredoxin NADP+-Reductase 0.
025u (Spinach) Ferredoxin (Clostridium, 5 μtahastoyrianum) Hofphatidylcholine 2In9 Ferrous Sulfate 1 fnM Final Capacity
Shake at 1 ytl at 30°C for 1 hour; react from scratch. Next, add 50 µli of 5N-NaOH and adjust pH% Mf to 41 m, then HPLC (column: Waters Radial Pack Cartridge C18, elution strip 14
:27L% acetonitrile 10.1%H3PO4, T
TF2 A (pH3,2)) Cytochrome P-45
0 is 5mura et al.'s method (, r, Biol, Che
It was identified by the reduced CO difference spectrum according to CN, 33-co1.2370, (1964).

Further, cytochrome P-450 was quantified according to the following formula.

Cytochrome P450 (nmolAl) = (0
, D45G -0, D490)
63086) and a transformed strain using the vector plasmid (Streptomyces lividans 5ANK60587
), but in the strain transformed with plasmid pHYO1, converting enzyme activity appears, and at the same time, cytochrome P-450 production is observed. In the plasmid-deleted strain, the production of cytochrome P-450 along with the converting enzyme activity was no longer observed, and by transforming the strain with plasmid pHYO1 or plasmid pHY○21, the converting enzyme activity reappeared, and at the same time cytochrome P-450 was also produced. will be done.

Plasmid pH with DNA fragments with different insertion directions
Both YO1 and pHYO 21 are originally ML-236B
Does not have the ability to convert Na to O8-514,
Furthermore, the ability to produce cytochrome P-450 at the same time as nuclear transmutation ability can be imparted to Streptomyces lividans 5ANK63086 or Streptomyces lividans 416P-7, which are hosts with extremely low ability even if they are present. Insert DNA contained in DNA
This shows that the P-450 gene, which has hydroxylase activity in its fragment, was cloned with the promoter region.

Area studies plasmids pHYO 21, pHYO 22 and p
Streptomyces-lividans 416 by HYO23
Transformed strains of P-7 (RTF-258, RTF-286
and RTF”-288), rML-23 such as Streptomyces lividans 5ANK60587 as a control method.
Preparation method of cell-free extract for "measurement of hydroxylase activity converting to 6BNaiC8-514" and "measurement of cytochrome P-450", rML-236BNaiC8-51
"Measurement of hydroxylase activity converting to 4" and "Measurement of cytochrome P-450" were carried out according to Test Example 2.

Figure 7 shows that cytochrome P-450 raw rice contains at least a 5phl fragment of about 1.5 kb generated by 5phI digestion and the vector of Bgt[I/MboI and the Shinto University DN.
A! ! It is shown that a DNA fragment of approximately 2.9 kb from the fr piece joining site to the Mlul cleavage site is required. That is, the cytochrome P-450 gene is contained in the plasmid p
Insert DNA fragment of HYO 23 and Bgt[/Mb o
Approximately 2.9 k from the l connection site to the Mtul cleavage site
This means that it exists in b. However, the host
It is thought that approximately 7.1 kb of the inserted DNA fragment of pHYO 21 is required to confer sufficient conversion activity from -236BNa to C8-514.

Plasmid PIJ 702 (Katz et al,
,,L Gen.

Microbiol, 129.2703 (1983)
tyrosinase gene (mal genea) present on
) is not expressed in all 7 Tredetomycetes. Therefore, the plasmid pMEL 18 was designed so that it can be cloned using melanin production as an indicator even in Streptomyces that cannot express the mal gene.
e was constructed. As a host Streptomyces that cannot express the mal gene of PIJ702, Streptomyces
Ji.

-Monjinensis [16]-8・5ANK61185(
Hereinafter, [16]-8・5ANK61185 strain) (FERM ap-t 140)
◎The msl gins of PIJ702 was injected into the host (16)-8-SANK61185 strain using
The DNA fragment that confers the ability to express e1gene i can be isolated from the chromosomal DNA of all Streptomyces, but here we isolated it from the cultured mycelium of Streptomyces nis p.
ur method (J, Mo1. Biol,, supra.

208 (1961)) was used as exogenous DNA.

5 μ2 of Streptomyces nisphi EiANK61184 DNA and 1 μ2 of plasmid P engineering J702 were mixed, and then 1/3 volume of a 4-fold concentration restriction enzyme reaction solution and 9 u% of a restriction enzyme Spk enzyme were added.

After culturing at 37°C for 2 hours to completely cleave DNA1, the mixture was heated at 70°C for 10 minutes to inactivate the restriction enzyme. To this sample, 1/10 volume of 3M sulfur-IJium acetate was added and stirred, and then 25 times the volume of ethanol cooled at -20°C was added, followed by cooling at -70°C for 10 to 20 minutes. Centrifuge this sample in a centrifuge, discard the supernatant, and precipitate the DNA.
The tL was washed with ethanol at -20°C. Next, it was dried in a vacuum, dissolved in sterile distilled water, and prepared to a 100-fold concentration using gauze reaction solution (66 Off 1M).

66ff1M MgCl2 ・H2O, I Go jn
M DTT, 1.1 mM ATP, pH 7,6
) was added in an amount of 179 volumes. Furthermore, T4 DNA ligase was added to this, and after incubating at 14°C for 16 hours, it was incubated at 65°C.
The gauze was inactivated by heating for 10 minutes. This was used as a sample for transformation. Using the transformation sample (
18)-II・5ANK61185 strain was transformed as follows. That is, put 20 d of aGcy medium in a 50 ute flask with a branch and add 06]-8.5ANK.
After inoculating the mycelium of the 61185 strain, it was incubated at 24-28℃ for 7 days.
Incubation was on a reciprocating shaker at 120 rpm for 2 hours. This was used as a seed and inoculated at a 5% amount into a 500-ag capacity Sakaro flask with 1 medium of GGC7 medium and cultured on a reciprocating shaker at 24 to 28°C for 24 hours. From this culture solution, obtain a pellet of the odontoid body by low-speed centrifugation.

This is 20 rttl P medium (320mMg sugar, 25mM TES buffer, ?Off1M saline, 10
After washing and suspending in 20mM MgCl2, 20mM Mc4c12). The bacterial cell pellet obtained by centrifugation was transferred to 20a again.
e suspended in P medium.

40η/I for this bacterial cell suspension! Add 1 me of tl concentration lysozyme solution and heat at 28°C for 1 hour [1B]-
Protoplasts of 8.5ANK 611115 strain were generated. The mixture of protoplasts and undissolved mycelia was naturally filtered through a glass filter (3G3) to obtain a protoplast solution containing a large amount of protoplasts, which was centrifuged at low speed and the pellet was resuspended in P medium. This operation was repeated three times and thoroughly washed to prepare a protoplast solution containing the desired number of protoplasts.

Transformation was performed by the method described in Example 1 using the previously prepared transformation sample. The protoplasts that had undergone the transformation procedure were appropriately suspended and spread on an R2MP regeneration medium (medium composition is described in Example 1). R2M
P Smeared on an agar plate and cultured at 28°C for 20 hours.
A 3 ml layer of soft agar R5 medium (medium composition is described in Example 1) to which thiopeptin was added to give a final concentration of 50 μl/me was layered on a 2MP agar plate. After Jl.

When the agar plate was continued to be cultured at 28°C, growth of 300 transformants showing resistance to thiopeptin was observed. Among them, seven strains that produced melanin pigment were recognized. Plasmids isolated from these strains harbored DNA fragments of 130 to 1540 base pairs (J) and were named npMIlcL18 to pMEL24, respectively. Furthermore, these plasmids pMEL18 to pME
Up to L24 is the host (:163-+1・5ANK 61
It was confirmed by re-transformation that the 185 strain was endowed with melanin pigment production.

Of these seven plasmids, pMEL 18 is 13
01) Since it has a DNA fragment of p, and no restriction enzyme recognition site for Bgl l or Sacl is found in this 130 bp fragment, P engineering J702 should be used as a cloning vector using this site. It can also be used in hosts that cannot. pure plasmid pM
Culture for extraction and production of EL1g was carried out as follows using a transgenic mutant (MEL 18 strain) harboring pME118.

Medium composition: glucose 0.4%, malt extract 1.0
% and yeast extract 0.4% and thiopeptin 25%
20at of medium added to μg/me; 150
After inoculating the mycelium of MEL18 strain into a flask with a branch, the mixture was heated at 24 to 28°C for about 72 hours at 120 rpm.
Cultured on a reciprocating shaker. Next, the composition of the medium for mycelium recovery is glycerol 0.4%, casamino acid 0.4%, yeast extract 0.05%, malt extract 0.1%, Mg5Oa.
0.1%, CaCl2-2H200.01%,
KH2P04G, 2% and Na2HPO4・12H2
00.8 ti (adjusted to pH 7.2) and added thiopeptin to 25 μg/tttl.
Place it in a 0ml 500IIL Yosaka flask and inoculate it with the suspension of the above seed culture in an amount equivalent to 1 to 5% of the culture medium.
Cultured on a reciprocating shaker for 24-48 hours at 24-28°C.

This culture solution is centrifuged at low speed (eg 10.GOO5').

The mycelium was removed at 4° C. for 20 minutes, and the supernatant was removed with a slant to obtain mycelium pellet 7. Plasmid pMIcL18
Extraction and purification of the mycelium pellet was carried out based on the method described in Example 3 using the resuspended mycelial pellet, and a pure plasmid pMEL 187i was obtained.

The size of the inserted DNA fragment in the recombinant plasmid was calculated by agarose gel electrophoresis. That is, 0.5 μg of one recombinant plasmid was treated with the restriction enzyme Sph.
By cutting with l, two bands were generated: a linearized 5 and 8 kilobase fragment of P engineering and T702 used as a vector plasmid, and an inserted DNA fragment of 1301)p. Hlnd of lambda DNA as a molecular weight marker
[[Hae of cut fragment or φX174 DNA ■
Using cut fragments.

Electrophoresis was carried out using agarose gel with different concentrations of 0.8%, 1.2%, and 2% depending on the size of the inserted DNA fragment, and the size of the inserted DNA fragment was determined from the mobility of the molecular weight marker.

The lasmid pMEL+8 thus obtained was Streptomyces lividans SANK 63086.
introduced into Streptomyces lividans S.A.N.
Deposited as K80587 (Feikoken=-V No. 9
No. 168).

[Brief explanation of drawings]

Figure 1 shows the C8-
Approximately 7.1 kb of DNA containing the gene for cytochrome P-450 with hydroxylase activity converting to 514
This is a restriction enzyme cleavage map of the fragment. In the figure, Sa is 5aC1, 3 is KpnI, P is Pstl
. M is Mlul, Sp is 5phl, X is Xhol, II:
(EωR) and Be indicate the cutting point due to Bcl (2). The numbers indicate the distance between each restriction enzyme cleavage position and are expressed in Kb. FIG. 2 is a restriction enzyme cleavage map of the recombinant plasmid pHYO1. The numbers represent Ba present in pMKLl8 used as the vector plasmid (represented by a thin line).
0IHl Each restriction enzyme when the cutting point is the coordinate point
Indicates the cutting position. The shaded area is plasmid pHYO2 and plasmid pH
It shows the homologous region with the inserted DNA fragment of YO21. FIG. 3 is a restriction enzyme cleavage map of the recombinant plasmid pHYO2. The numbers represent the cleavage positions of each restriction enzyme when the BamW I cleavage point on the DNA fragment derived from the vector plasmid (represented by a thin line) is taken as the coordinate point. The shaded area is insertion D of plasmid pHYO 1.
It represents the homologous region with the NA fragment. FIG. 4 is a restriction enzyme cleavage map of recombinant Grasmid pHYO 21. The numbers represent the cleavage positions of each restriction enzyme when the BamHI cleavage point on the DNA fragment derived from the vector Grasmid (represented by a thin line) is taken as the coordinate point. This plasmid is plasmid pHYO2s
The approximately 2.4 Kb DNA fragment generated by acl digestion was removed. The shaded area is plasmid pHYO2
It is the same as the shaded part. FIG. 5 is a restriction enzyme cleavage map of recombinant Grasmid pHYO 22. The numbers represent the cleavage position of each restriction enzyme when the BamHJ cleavage point on the DNA fragment derived from the vector grass band (represented by a thin line) is taken as the coordinate point. This grass band is obtained by removing an approximately 1.5 Kb DNA fragment generated by sphl digestion of plasmid pHYO 21. FIG. 6 is a restriction enzyme cleavage map of recombinant plasmid pHY023. The numbers represent the cleavage positions of each restriction enzyme when the coordinate point is the Bam) (I cut point) on the DNA fragment derived from the vector Grasmid (represented by a thin line). Plasmid pHYO21 M
Approximately 3.6 Kb and approximately 0.9 Kb of DNA generated by tuI digestion
The A fragment has been removed. Figure 7 shows the inserted DNA fragment of approximately 7. This figure shows the results of an examination of the localization site of the cytochrome P-450 gene in IKb. Converting enzyme activity and cytochrome P-45 in host Streftbases lividans transformed by each Grasmid
0 production is shown. In the figure, the black line represents the vector DNA fragment, the white line represents the inserted DNA fragment, and the dotted line represents the region of the removed DNA fragment. Patent questioner Sankyo Co., Ltd.

Claims (1)

[Scope of Claims] 1. A DNA fragment containing the actinomycete cytochrome P-450 gene and capable of imparting hydroxylation activity to a host. 2. DNA containing the P-450 gene and capable of imparting hydroxylation activity to the host is ML-236B sodium salt (ML-2
36BNa) to CS-514, which is an approximately 7.1 kb DNA fragment derived from the chromosome of actinomycetes and has a restriction enzyme cleavage map shown in the figure below. (BglII/Mbol) ▲ Contains mathematical formulas, chemical formulas, tables, etc. ▼ 3. DN with a region containing the P-450 gene of approximately 2.9 kb
A DNA fragment having the restriction enzyme cleavage map shown in the figure below. (BglII/Mbol) ▲ Contains mathematical formulas, chemical formulas, tables, etc. ▼ 4. DNA according to claim 1, where the DNA is a plasmid pHYO1, pHYO2 or pHYO21.
. 5. DNA which is plasmid pHYO23 containing the DNA fragment according to claim 3. 6. Plasmid pHYO1, pHYO2 as DNA,
A microorganism containing pHYO21 or pHYO23.