JPS62155081A - Novel microorganism and production of biotin by fermentation with said microorganism - Google Patents

Abstract

PURPOSE:To stably produce biotin in high production efficiency, by introducing a specific plasmid to a mutant of E.coli inert to feedback inhibition with biotin and culturing the obtained microorganism. CONSTITUTION:A biotin-producing wild strain of Escherichia coli (EC) is mutated to obtain a biotin-requiring strain (BR). Separately, another EC strain is mutated to obtain a mutant strain (DRK) free from feedback inhibition with biotin. A DNA containing biotin operon is separated from the EC wild strain. The DNA is integrated in a vector DNA and the obtained recombinant DNA is introduced into the BR strain to obtain a biotin-producing microbial strain, from which a recombinant DNA plasmid is separated. The DNA plasmid corresponds to <=3760 base pairs downstream of a translation initiation point of a DNA carrying a genetic information of an enzyme converting a desthion biotin of a terminal of biotin operon to biotin. The recombinant DNA plasmid is introduced into a DRK strain and the resultant EC-DRK-332 strain is cultured in a medium to accumulate biotin.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field 1] The present invention relates to a novel microorganism and a method for producing biotin using the same.

[Prior Art] Biotin is a type of vitamin that is important for animals, plants, and microorganisms.

Conventionally, known methods for producing biotin using microorganisms include methods using microorganisms of the genus Bacillus, Chromobacterium, Schutomonas, Arthrobacterium, and the like. Furthermore, a method has also been proposed in which these wild strains are artificially mutated to impart biotin-producing ability. (Unexamined Japanese Patent Publication No. 58-60996 wf&) [Problems to be Solved by the Invention] However, when trying to produce biotin using microorganisms, wild strains have a strong feedback inhibition mechanism by biotin (Y, Izumi, In,Ogata,Ad
v, Appl, former crobia1. Vol, 22, 1
55-157.1977) Biotin is produced in very small amounts. Furthermore, even with methods using mutant strains, the amount produced was not always satisfactory.

The name of the present invention focuses on breeding through genetic manipulation, which is different from these methods, and as a result of intensive research to obtain microorganisms with excellent biotin-producing ability, biotin can be produced by artificially mutating a wild strain of Escherichia coli. We obtained a mutant strain in which the feedback inhibition by
A was isolated, and then the DNA was integrated into vector DNA to obtain a recombinant DNA plasmid, and the recombinant DNA plasmid was successfully introduced into the mutant strain. After discovering that it had production capacity, I applied for a patent (patent application filed in 1986).
-42928), but when the above method was used, the recombinant DNA plasmid was in an unstable state where gene deletion sometimes occurred.

[Means for Solving the Problems 1] In view of the above circumstances, the present inventors conducted further research to solve these shortcomings, and as a result, one end of the wild strain of Escherichia coli that has biotin-producing ability was obtained. The biotin operon is extracted so that the number of base pairs is below the specified number, and then a recombinant DNA plasmid is obtained in which the biotin operon is integrated into vector DNA, and the recombinant DNA plasmid is artificially mutated into a wild strain of Escherichia coli. In addition to successfully introducing biotin into a mutant strain in which feedback inhibition by biotin was released by
The microorganism thus obtained is stable without the above-mentioned drawbacks,
It was discovered that it has an excellent ability to produce biotin, and based on these findings, the present invention was completed.

That is, the present invention provides (1) translation of a biotin operon obtained from Escherichia coli having the ability to produce biotin, in which one end of the biotin operon carries genetic information for an enzyme involved in the conversion of desthiobiotin to biotin; (2) a microorganism obtained by incorporating a recombinant DNA plasmid in which a biotin operon, which is 3760 base pairs or less downstream from the starting point, into the vector DNA, is contained in an Escherichia coli mutant strain in which feedback inhibition by biotin has been released; and (2) the microorganism This method of producing biotin is characterized by culturing and collecting biotin produced and accumulated in the medium.

The present invention will be explained in detail below.

The biotin operon referred to in the present invention refers to A, B, F, and C shown in FIG. 1, which are involved in biotin biosynthesis.

This refers to a DNA region that includes all the control regions necessary to express the D gene and those genes that exist between A and 8. In addition, the above A, B, F, and C.

D represents the DNA region that carries the genetic information of the enzymes listed below. (J, Mo1. Biol, Vol, 56.
53-62゜A nia, 8-diaminopelargonic acid synthetase B: Enzyme involved in the conversion of desthiobiotin to biotin F nia-keto-8-aminopelargonic acid synthetase C: Pimeryl CoA synthetase D: Desthiobiotin synthetase to 7′′.

First, a mutation is induced in an Escherichia coli wild strain (for example, Escherichia coli JA221) having biotin-producing ability to obtain a biotin-requiring strain (hereinafter referred to as a BR mutant strain). Next, mutations are induced in the Escherichia coli wild strain (for example, Escherichia coli W3110) to obtain a mutant strain in which feedback inhibition by biotin has been released (hereinafter referred to as a DRK mutant strain). Mutation can be induced using conventional mutagenesis treatments, such as N-methyl-N
This can be carried out by treatment with a mutagenic agent such as '-nitro-N-nitrosoguanidine. To obtain the BR mutant strain, the cells obtained by mutagenesis treatment are appropriately diluted with a buffer solution, and then cultured on a minimal medium agar plate containing biotin, and an appropriate number of colonies (approximately 50 to 200 (IW)) appear. Select the prepared plate, replicate it on a biotin-free minimal medium agar plate, culture it, compare the replica plate with the master plate, and isolate colonies that do not grow on the replica plate from the master plate.

Examples of the BR mutant strain thus obtained include Escherichia coli BR-4. Since this BR mutant strain cannot grow even on minimal medium supplemented with desthiobiotin,
This is caused by a deficiency in the enzyme involved in converting desthiobiotin to biotin.

In addition, known BR mutant strains include, for example, J, Bact
eriol, , Vol, 112, 830-839
R876 strain, R874 strain, R87 described in (1972)
3 strains, R877 strains, and R875 strains (C gene, F gene, respectively)
gene, A gene, D gene, B gene deleted strain)
can be given.

On the other hand, to obtain the DRK mutant strain, the bacterial cells obtained by mutagenesis treatment were appropriately diluted with a buffer solution, and the B.
R mutant strain and 2.3.5- A biotin-free minimal medium agar plate containing triphenyltetrazolium chloride was cultured on a plate prepared by overlaying a certain amount of biotin-free minimal agar medium. This is done by fishing out and isolating small colonies whose lower layer shows a pink zone. This principle is based on the fact that the DRK mutant strain, in which the feedback inhibition by biotin has been released, produces a large amount of biotin and helps the growth of the BR mutant strain in the lower layer, and as it grows, 2,3.5-triphenyltetrazolium chloride is reduced. The reason is that it exhibits a pink zone. Therefore, such a phenomenon is not observed in the wild strain in which no mutation has been induced. An example of the DRK mutant strain obtained in this manner is Escherichia coli DRK-332 (Kouken Bacterial Collection No. 2989).

The fact that this DRK-332 strain is not an operator mutation is that
After isolating and purifying the biotin operon by the method described below, the base sequence of the regulatory region for gene expression, including its operator, was extracted by chemical decomposition method (Methods in Enzyme).
mology Vol, 65.499-580.198
0) or dideoxynucleotide enzymatic method (Metho
ds in Enzymology Vol, 101.
20-78.1983), and can be confirmed by comparing with the same region of the wild strain. In other words, if the nucleotide sequence is exactly the same as the same region in the wild strain, it is presumed that it is not an operator mutation but probably a repressor mutation.

Next, in order to isolate and purify DNA containing the biotin operon (hereinafter referred to as chromosomal DNA) from the wild strain, for example, Biochim, Biophys, Acta V
It can be carried out according to a conventional method such as the phenol method described in 1 (163).

Next, the obtained chromosomal DNA is incorporated into vector DNA to prepare recombinant DNA. Chromosomal DNA vector D
Incorporation into NA can be carried out according to conventional methods. For example, this can be carried out by cutting chromosomal DNA and vector DNA with a restriction endonuclease to prepare a chromosomal DNA fragment and a vector DNA fragment, and then treating a mixture of the two with DNA ligase. The vectors used here include the pBR322 plasmid, which uses Escherichia coli as a host, and colicin (Col
) E1 plasmid, lambda phage, and other commonly used plasmids can be employed. In particular, pBR322 plasmid is preferably used. Examples of restriction endonucleases include old ndl11. ．． EcoRT, PstI
, BamHI, etc., but many restriction endonucleases can be used in this experiment as long as the manipulation of the restriction endonuclease is shallow, that is, the cleavage of DNA1 is made to stop at partial cleavage.

It is also possible to use two types of restriction endonucleases together.

FIG. 1 shows a restriction endonuclease cleavage map of the biotin operon. As the restriction endonucleases used in the present invention, those shown in the diagram are particularly preferably used.

As the DNA ligase, DNA ligase derived from T4 phage is preferably used.

Next, the recombinant DNA obtained in the above manner is subjected to a conventional method such as [Molec, Gen, Genet, Vol.
124.1-10 (1973)] into a BR mutant strain (e.g. Escherichia coli BR-4) and cultured on a minimal medium agar plate without the addition of biotin. A bacterial strain isolated from fish that has the ability to produce biotin (i.e., a biotin operon or a recombinant DNA into which a part of the biotin operon has been incorporated)
A strain containing the A plasmid that has recovered biotin auxotrophy) is collected.

Next, a recombinant DNA plasmid is isolated from the recombinant DNA plasmid-containing bacterial strain obtained by the above method.

Isolation of recombinant DNA plasmids can be performed according to conventional methods. For example, Nucleic acid rese
arch, Vol, 7.1513-1523 (19
Examples include the alkaline extraction method described in 79).

It can be confirmed as follows that the recombinant DNA plasmid thus obtained contains the biotin operon.

That is, this is confirmed by the fact that the biotin requirement can be completely restored by introducing a wood recombinant DNA plasmid into an Escherichia coli BR mutant strain deficient in each biotin biosynthetic enzyme activity. Depending on the type of restriction endonuclease used to obtain the recombinant DNA plasmid, the entire biotin operon may not be included, that is, some genes may be missing, when the treatment is complete. In such cases, reconstitution from a customary recombinant DNA plasmid obtained using different restriction endonucleases can be carried out, resulting in a recombinant DNA plasmid containing the biotin operon.

Depending on the type of restriction endonuclease used here, one end of the biotin operon carries the genetic information for the enzyme involved in the conversion of desthiobiotin to biotin.
There are cases where the length exceeds 3760 base pairs downstream from the NA translation start point. In this case, because the gene region exists downstream from that site, instability of the recombinant DNA plasmid is often observed, such as gene deletion occurring in the obtained recombinant DNA plasmid. must be deleted. The translation initiation site of the DNA that carries the genetic information for the enzyme involved in the conversion of desthiobiotin to biotin is the restriction endonuclease Nco shown in Figure 1.
ATG of I recognition site 5°-CCATGG-3° (C: cytosine, A: adenine, T: thymine, Gnigeanine)
(Nature Vol.

276.689-6'14.0178).

The region limited by this patent may be 3760 base pairs or less from this ATG, but it is within the biotin operon, that is, the first
It goes without saying that it should not be below the Da gene region shown in the figure. In other words, one end of the biotin operon is 3603 base pairs or more and 3760 base pairs or less downstream from the translation start point of the DNA that carries genetic information for the enzyme involved in converting desthiobiotin to biotin, and particularly preferably 3664 base pairs. The number of base pairs is greater than or equal to 3760 base pairs.

To delete the unnecessary gene region downstream of the D gene in the recombinant DNA plasmid, the recombinant DNA is cut with an appropriate restriction endonuclease, then partially digested with the endonuclease starting from the cut point, and after religation, the BR mutant strain is extracted. can be obtained by introducing it into

By introducing the recombinant DNA plasmid obtained as described above into the DRK mutant strain described above, a DRK mutant strain containing the recombinant DNA plasmid can be prepared.

Depending on the characteristics of the vector, the DRK mutant strain containing the recombinant DNA plasmid can be obtained as colonies that appear by culturing on an agar plate that selectively grows bacterial clones containing the recombinant DNA plasmid.

Examples of the thus obtained recombinant DNA plasmid-containing DRK mutant strain, that is, the novel microorganism of the present invention, include Escherichia coli DRK-332 [pKHN31] (
Koukoken Bacteria Serial No. 853 et al.).

If the microorganism of the present invention obtained as described above is cultured, a significant amount of biotin will be produced and accumulated in the culture solution.

As the medium used for culturing the microorganism according to the present invention, either a synthetic medium containing a carbon source, a nitrogen source, and an inorganic substance, or a natural medium can be used. As the carbon source, carbohydrates such as glucose, glycerin, fructose, sucrose, maltose, mannose, starch, starch hydrolyzate, and molasses can be used, and the amount used is 0.
About 5 to 5.0% is preferable. Nitrogen sources include various inorganic and organic ammonium salts such as ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium carbonate, and ammonium acetate, or meat extract, yeast extract, cornstarch liquor, casein hydrolyzate, and defatted soybean meal. Alternatively, natural organic nitrogen sources such as its digested material can be used, and the amount used is 0.5~
About 2.0% is preferable.

Many natural organic nitrogen sources can be both nitrogen and carbon sources.

Inorganic substances include potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, sodium chloride,
Ferrous sulfate, calcium chloride, zinc chloride, steel sulfate, manganese chloride, cobalt chloride, ammonium molybdate, boric acid, etc. can be used, and the amount used is preferably about 0.005 to 0.5%.

In addition, if the created microorganism is endowed with antibacterial drug resistance, contamination with contaminating bacteria can be prevented by adding the corresponding antibacterial drug to the culture medium.

The culture is preferably carried out under aerobic conditions such as shaking culture or aerated agitation culture. Cultivation R? The H degree is preferably 25 to 37°C, the PO of the medium during culture is desirably maintained near neutrality, and the culture period is usually sufficient for about 16 to 48 hours. After completion of the culture, biotin can be extracted and purified from the culture solution using general extraction and purification methods from natural products, making use of the various properties of biotin. Zunawara,
Remove the bacterial cells from the culture and adsorb them onto activated carbon.
Biotin can be obtained by elution and purification with an ion exchange resin, or by directly treating and purifying the culture filtrate with an ion exchange resin, followed by recrystallization from water or alcohol.

[Example] Next, the present invention will be explained in detail with reference to Examples.
The present invention is not limited to these examples.

Example 1 (1) Adjustment of D RK mutant strain Wild strain Escherichia co! having biotin-producing ability. , I
W3110 (IFO12713) WoL-medium (peptone 10 gz"l.

Yeast extract 5g/1. Glucose Ig/l, sodium chloride 5g/l (L, pH adjusted to 7.2) at 37°C
After culturing with shaking for 3 hours and observing the bacterial cells in the logarithmic growth phase,
This was mixed with a TM buffer (Tris-
The cells were suspended in 0.61% base, 0.5% maleic acid, adjusted to pH 6.0) and allowed to stand at 37° C. for 30 minutes to perform mutation treatment. After testing the bacterial cells, the suspension was tested at 105 cells.
l/rrL BR mutant strain Escherichia coυBR-
20 mL biotin-free minimal medium agar plates containing 4 and 50 ug/me of 2.3.5-triphenyltetrazolium chloride (glucose 5 g/4. - Potassium hydrogen Ig/l, magnesium sulfate heptahydrate 0.18/l, vitamin-free casamino acids 4g/l
, agar 15g/l! , ) was plated on a regular sized petri dish overlaid with 15+++e biotin-free minimal agar medium so that about 50 to 200 colonies would appear. D
RK mutant Escherichia coli DRK-332 (@”I
Koken Bibori No. 8939) was obtained.

(2) Escherichia coli v3110 with chromosomal DNA regulation biotin production ability
500 mQ, cultured for 3 hours at 37°C in Noshi medium for 1 hour, washed the cells in the logarithmic growth phase, extracted and purified by the usual DNA extraction method using the phenol method, and obtained 1.8 mg.
Chromosomal DNA was obtained.

(3) Preparation of vector DNA pBR322 plasmid DNA having ampicillin resistance and tetracycline resistance was prepared as follows.

First, one of the 112 strains of Escherichia coli carrying pBR322 as a plasmid was injected with Ancipillin 507.
37°C in L-medium 10100O supplemented with 1g/mL
150 Ltg/ml of chloramphenicol was added at the logarithmic growth 91a phase, and cultured overnight in a colander. This operation allows a large amount of plasmid DNA to be produced within the cells.

After adding chloramphenicol, the cells were collected for 15 hours, lysed with lysotium and sodium dodecyl sulfate, and then centrifuged at 30,000 x g for 2 hours to obtain a supernatant. After concentrating plasmid DNA from this supernatant, it was treated with ribonuclease for 2 hours at 37°C, and then a final fraction of 450 μg of pBR322 plasmid DNA was collected by cesium chloride ethidium bromide equilibrium density gradient centrifugation.

(4) Inserting the chromosomal DNA fragment into the vector Take 2 μg of the DNA obtained in (2) and inject it with restriction endonuclease pst.
I was added and reacted at 30°C for 3 hours to completely cleave. In addition, 1.5 μg of the vector DNA prepared in (3) was added to pstI
completely severed. Both reaction solutions were heat-treated at 65°C for 5 minutes, then mixed, and DNA was ligated with T4 phage-derived DNA ligase at 15°C for 16 hours in the presence of ATP (adenosine triphosphate) and dithiothreitol.
A ligation reaction of chain A was performed.

Similarly, 2 μg of the chromosomal DNA obtained in (2) was simultaneously reacted with two restriction endonucleases, EcoRI and BamHI, at 37°C for 3 hours. After heat treatment at 65°C for 5 minutes, the vector DNA was completely digested with EcoRI and BamHI. The mixture was mixed and a DNA ligase reaction was performed at 15° C. for 16 hours. After each reaction was heat treated at 65° C. for 5 minutes, twice the volume of ethanol was added to the reaction solution to precipitate and collect the DNA after the ligation reaction.

(5) Transformation with recombinant DNA containing the biotin operon or a part of the biotin operon Escherichia coli BR-4, a biotin-auxotrophic strain mutagenized from Escherichia coli JA221, was inoculated into 10 mN of L-medium and incubated at 37°C.
After culturing with shaking for 4 hours and growing to the mid-logarithmic phase, the bacteria were collected, washed twice with Tris buffer (50mM, p) 17°0) containing calcium chloride 50m)4, and then washed twice with the same buffer. It was resuspended in 1 cup.

The recombinant DNA solution obtained using pstl in (4) was added to this 'P suspension at 0.2 mQ, kept at 0°C for 30 minutes, and immediately subjected to heat shock at 42°C for 2 minutes.
NA was taken into the cells. Then, a certain amount of each cell suspension (vA solution) was taken into fresh L-medium and incubated at 37°C.
Static culture was performed for hours. After each bacterial cell was collected, the resuspension was spread on a minimal medium agar plate and cultured at 37°C for 2 days. The resulting colony was picked and transformed to obtain Escherichia coli BR-4 [pOFT53].

Recombinant DNA obtained using the two restriction endonucleases EcoRI and Bam in (4) in exactly the same way
Escherichia coli BR transformed with Ai8' solution
-4[pBDR72] was obtained.

The obtained transformed strain Escherichia coli BR-4 [pO
FT53] and Escherichia coli BR-4 [pBD
R72] were cultured in 100 ml of L-medium, and (
Chloramphenicol treatment was performed in the same manner as in 3).

After collecting the bacterial cells, the alkaline method (Nucleic Ac1d
sRe5earch, E+ 1513-1523
, 1979), Escherichia coli BR-4 [
pOFT53] to recombinant DNA plasmid pOFT5
3 to Escherichia coli BR-4 [pBDR? A large amount of recombinant plasmid pBDR72 was obtained from [2].

Recombinant DNA plasmids pOFT53 and pBDR7
After treating 2 with various restriction endonucleases, the restriction endonuclease cleavage pattern was compared with the restriction endonuclease cleavage map of the biotin operon in Figure 1, and it was found that the recombinant DNA plasmid pOFT53 has two psTIs of the biotin operon in Figure 1. Now, the clean area is about 53
80 base pairs, also recombinant DNA plasmid pBDR7
2 is Bam1lI, E of the biotin operon in Figure 1.
It contained a region of approximately 5450 base pairs that is separated by coRI.

(6) Construction of a recombinant DNA plasmid by deleting unnecessary gene regions in recombinant DNA plasmid pBDR72 Recombinant DNA plasmid pBDR-7 obtained in (5)
Take 2 μg of 2, complete cleavage with restriction endonuclease EcoRI, partially digest with endonuclease Ba131 starting from the cleavage point, completely stop the reaction with phenol treatment, and precipitate DNAΔ by adding 2 volumes of ethanol. I picked it up. Hindl was added to the end of the obtained DNA fragment.
The linker was ligated by the action of DNA ligase derived from T4 phage. Next, restriction endonucleases Hindll and Bam were added to the reaction solution to completely cleave the reaction solution, and then subjected to agarose electrophoresis (agarose 1%, 70V) and stained with ethidium bromide.
A gel piece containing a DNA fragment corresponding to Kb was cut out under ultraviolet irradiation, this gel was placed in a dialysis tube, electrophoresis was performed again, and DNA was extracted from the gel. After extraction, remaining agarose was removed by phenol treatment, and then 2 volumes of ethanol was added to submerge the DNA and collect it.

The obtained DN distributed from about 4.5 Kb to about 5.2 Kb
A fragment and two other restriction endonucleases, old nd1
11. Approximately 4.4. OKb pBR
322 vector DNA fragment was mixed, and a ligation reaction was performed using DNA ligase derived from T4 phage.

Next, after heat treatment at 65° C. for 5 minutes, twice the volume of ethanol was added to the reaction solution, and the DNA after the completion of the ligation reaction was collected by precipitation.

゛Recombinant DNA with DNA fragments of various sizes obtained
A to BR mutant Escherichia coli BR-4 strain (5)
Transformation was performed in the same manner as described above, and colonies produced on a minimal agar plate without addition of biotin were harvested.

To check the stability of the recombinant ffNA plasmid, various colonies obtained were treated with ampicillin (50 μg/drill).
After incubating three times on a biotin-free minimal agar medium, extract the recombinant DNA plasmid from each colony using the same method as in (5), and examine the restriction endonuclease cleavage pattern using an appropriate restriction endonuclease. Table 1 shows the results of examining the stability of the recombinant DNA plasmid, including the presence or absence of the recombinant DNA plasmid containing the deleted gene region.

Table 1 shows that one end of the biotin operon contained in the recombinant DNA plasmid is the translation initiation site of the DNA that carries the genetic information for the enzyme involved in converting desthiobiotin to biotin (i of the restriction endonuclease Nco I). ,l
Bg1 site 5°-CCA T G G-3' AT
The distance (number of base pairs: bp) from
sin Enzymology Vol, 101.20
-78.1983). In addition, the recombinant DNA plasmid extracted from each colony was transformed into R877, a type of BR mutant strain (a strain lacking the D gene), using the same method as in (5), to examine whether biotin requirement was restored. The results were also shown at the same time. As is clear from the results, one end of the biotin operon is less than 3760 base pairs downstream from the translation start point of the DNA that carries the genetic information for the enzyme involved in the conversion of desthiobiotin to biotin. is essential for the stability of Furthermore, as a result of the restoration of biotin auxotrophy in the biotin auxotroph strain R877, one end of the biotin operon is located at least 3603 base pairs downstream from the translation start point of the DNA that carries the genetic information for the enzyme involved in the conversion of desthiobiotin to biotin. The length is preferably 3664 base pairs or more, especially considering the transcription termination point.

(Margin below) Table 1 (Margin below) 4′-d-1- It was stable, with no O2 gene deletion occurring.

×: Recombinant DNA with gene deletion after three tli passages
Plasmids were mixed.

1 ζ O' L no J and round stock B' + Recovery of nibiotin requirement was observed.

−: Recovery of biodin requirement was not observed.

(7) Construction of recombinant DNA plasmid containing biotin operon Transformant strain Escherichia coli BR- obtained in (5)
4 [pOFT53] and Escherichia coli BR-4 [pBDR91] obtained in (6) were each cultured in 100rnt of L-medium, and treated with chloramphenicol in the same manner as in (3). After observing the bacterial cells, the alkaline method (Nucleic Ac1ds Research,
Z, 1513-1523, 1979),
A large amount of recombinant DNA plasmid pOFT53 was obtained from Escherichia coli BR-4 [pOFT53], and a large amount of recombinant DNA plasmid pBDR91 was obtained from Escherichia coli BR-4 [pBDR')1].

2 μg of recombinant DNA plasmid pOFT53 was taken, and after complete cleavage with restriction endonuclease Kpn I, the reaction was stopped with phenol treatment, and 2 volumes of ethanol was added to precipitate and collect the DNA. The old ndllll linker is ligated to the end of the obtained DNA fragment using the action of T4 phage-derived DNA ligase. Next, restriction endonuclease old ndll [and NcoI] were added to the reaction solution to completely cleave the reaction solution, and then subjected to agarose electrophoresis (agarose 1%, 70 V) and stained with ethidium bromide.
A gel piece containing the A fragment was cut out under ultraviolet irradiation, this gel piece was placed in a dialysis tube, electrophoresis was performed again, and DNA was extracted from the gel. After extraction, remaining agarose was removed by phenol treatment, and then 2 volumes of ethanol was added to precipitate and collect the DNA. Separately, pBDR91 was cut with two restriction endonucleases, old nd III and Heal, and after agarose electrophoresis, a DNA fragment of approximately 3.7 Kb obtained by recovering from the gel in the same manner was further cut completely with restriction endonuclease l1indll. The resulting pBR322 hector DNA fragment was prepared.

The above three DNA fragments were mixed and a ligation reaction was performed using DNA ligase derived from T4 DNA phage.

Next, after heat treatment at 65° C. for 5 minutes, twice the volume of ethanol was added to the reaction solution, and the DNA after the completion of the ligation reaction was collected by precipitation.

The obtained recombinant DNA was transformed into the BR mutant Escherichia coli BR-4 strain in the same manner as in (5), and colonies generated on a minimal agar medium without addition of biotin, that is, Escherichia coli BR-4 [ pKHN31] was caught and its properties were investigated, and it was found to be ampicillin-rigid and tetracycline-sensitive.

This transformed strain Escherichia coli BR-4 [pKHN
31] by the same alkaline method as described above, and the obtained recombinant DNA plasmid pK
IIN31 was roughly transformed into a BR mutant strain deficient in each of the genes contained in the biotin operon described above in the same manner as in (5). The results are shown in Table 2.

As is clear from Table 2, the recombinant DNA plasmid P
Recovery of biotin auxotrophy was observed in all BR mutant strains transformed with K HN 31, indicating that the recombinant DNA plasmid pKHN31 constructed here contains a biotin operon.

(8) Transformation of the recombinant DNA plasmid containing the biotin operon into Escherichia coli DRK-332 (5)
Escherichia coli DRK332 was transformed using the same method as above, and ampicillin 5011 g/m! ! Isolate the resulting colonies on a single medium agar plate containing Ethie')
Here Ko') DRK-332[pKIIN31] (
? a Engineering? iJT I￥j? r'j35&/)th
No.) was obtained.

Example 2 (Medium composition) Glucose 5.0g Ammonium sulfate 4.0g Casamino acids (vitamin free) 4.0g Potassium dihydrogen phosphate 2.0g Potassium hydrogen phosphate
1.0g Magnesium sulfate heptahydrate 0
．． 1g ion exchange water 1000+
The strains shown in Table 3 were inoculated into a medium (pH 7.0) having the above composition and cultured with shaking at 37°C for 20 hours. After the culture is completed, the bacterial cells are removed by centrifugation, and the amount of biotin produced and accumulated in the culture solution is collected by Lactobacillus prandarum (Lactobacillus prandarum).
tbacillus plantarum, ATCC
8014) using the microbial quantitative method. The results are shown in Table 3.

Table 3 Example 3 (Medium composition) Glucose 5.0g Ammonium sulfate 5.0g Yeast extract
5.0g proteose pebtone (D
irco) 5.0 g sodium chloride
5.0g Potassium dihydrogen phosphate
4.0g Magnesium sulfate heptahydrate 1.
0 g ion exchange water 1000
The strains shown in Table 4 were inoculated into a medium (pl+7.o) having the above composition, and cultured with shaking at 37°C for 20 hours. After the culture was completed, the bacterial cells were removed by centrifugation, and the amount of biotin produced and accumulated during each culture was determined using the same microbial assay method as in Example 2. The results are shown in Table 4.

Table 4

[Brief explanation of drawings]

FIG. 1 is a drawing showing the five gene regions forming the biotin operon and the restriction endonuclease cleavage sites present in and near them. FIG. 2 is a diagram showing a method for constructing a recombinant DNA plasmid in which unnecessary gene regions of the recombinant DNA plasmid pBDR72 shown in (6) of Example 1 have been deleted. FIG. This is a drawing showing the method for constructing the recombinant DNA plasmid pKtlf131 shown in 7). Patent applicant F 1; Company Shiseido (21st blade~ ↓εcoRIt7Fm'j ↓8a131Q filling C-

Claims (3)

[Claims] (1) Escherichia coli (E
A biotin operon obtained from Scherichia coli, in which one end of the biotin operon is located 3,760 base pairs downstream from the translation start point of deoxyribonucleic acid (DNA), which carries the genetic information for the enzyme involved in the conversion of desthiobiotin to biotin. A microorganism in which a recombinant DNA plasmid in which a certain biotin operon is incorporated into vector DNA is contained in an Escherichia coli mutant strain in which feedback inhibition by biotin has been released. (2) One end of the biotin operon obtained from Escherichia coli, which has the ability to produce biotin, initiates the translation of deoxyribonucleic acid (DNA), which carries the genetic information for the enzyme involved in the conversion of desthiobiotin to biotin. A microorganism containing a recombinant DNA plasmid in which a biotin operon, which is 3760 base pairs or less downstream from the point, is incorporated into the vector DNA, is contained in an Escherichia coli mutant strain in which feedback inhibition by biotin has been released, and a microorganism is produced in the medium. A method for producing biotin, which comprises collecting accumulated biotin. (3) pB whose vector hosts Escherichia coli
The microorganism according to claim 1 or the production method according to claim 2, which is an R322 plasmid.