JPH04325086A - Transformant bacterium belonging to genus serratia and production of biotin using the same bacterium - Google Patents

Abstract

PURPOSE:To efficiently produce a transformant bacterium belonging to the genus Serratia and biotin by using the bacterium. CONSTITUTION:A DNA encoding biotin synthase derived from a bacterium belonging to the genus Serratia is taken out and the DNA integrated into a vector transferrable to the bacterium belonging to the genus Serratia to constitute a recombinant vector. The bacterium belonging to the genus Serratia is transformed with the recombinant vector to prepare a transformant bacterium belonging to the genus Serratia. The bacterium is aerobically cultured to efficiently produce biotin.

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a microorganism incorporating a biotin synthetase gene and a method for producing biotin using the microorganism, and more specifically relates to the reaction of a microorganism belonging to the genus Bacillus to convert desthiobiotin into biotin. The present invention relates to a method for producing biotin, which comprises culturing a microorganism belonging to the genus Serratia that has been transformed with a recombinant vector having DNA encoding an enzyme.

0002

BACKGROUND OF THE INVENTION Biotin is an essential vitamin for humans, animals and plants, and is important as a pharmaceutical or feed additive. Recently, the application of genetic recombination technology has been considered as a manufacturing technology.

[0003] Microorganisms belonging to the genus Escherichia, Bacillus, and Pseudomonas are known as hosts into which genes involved in biotin synthesis are incorporated (Japanese Unexamined Patent Application Publication No. 1989-1992).
1-149091, JP 62-155081, JP 62-275684, JP 63-44889).

On the other hand, as a gene involved in biotin synthesis, desthiobiotin (
An enzyme (hereinafter referred to as biotin synthetase) that is involved in the reaction of converting DTB (hereinafter referred to as DTB) into biotin is known (Japanese Patent Application Laid-Open No. 62-275684), and has been reported to have high activity ( Said Japanese Unexamined Patent Publication No. 1986-
No. 44889).

[0005] In addition, as an example of using a microorganism belonging to the genus Serratia as a host, a method for producing biotin by incorporating part or all of the gene involved in biotin synthesis into a microorganism belonging to the genus Serratia is also known. However, there has been no example of incorporating DNA encoding biotin synthetase from a microorganism belonging to another genus into a microorganism belonging to the genus Serratia.

[0006] Generally, it is difficult to predict that a recombinant microorganism incorporating the DNA of a microorganism belonging to another genus will express the DNA and that the expressed protein will exhibit high activity. This is because microorganisms of different genera often have low DNA homology.

[0007] When the DNA is DNA encoding biotin synthetase, the base sequences of microorganisms belonging to the genus Escherichia and Bacillus are already known.
However, when these are compared, the homology is calculated to be 32%, and the nucleotide sequences of biotin synthetase differ greatly between genera. This suggests that the properties of biotin synthetase produced and the reaction conditions for biotin synthetase also differ between genera.

[0008] Therefore, when DNA encoding biotin synthetase from a microorganism belonging to another genus is introduced into a microorganism, it is not necessarily possible to predict whether biotin synthetase will be expressed and whether the expressed protein will be able to exhibit high activity. It was difficult to do so.

[0009]

[Problems to be Solved by the Invention] The present inventors have conducted intensive research to obtain a microorganism with high biotin production ability that incorporates DNA encoding biotin synthetase derived from a microorganism of the genus Bacillus. A transformed microorganism belonging to the genus Serratia having high biotin-producing ability can be obtained by cutting out a coding DNA fragment from a microorganism belonging to the genus Bacillus and transferring it into a microorganism belonging to the genus Serratia using a vector incorporating the fragment. The present invention was completed based on this finding.

0010

[Means for Solving the Problems] Thus, according to the present invention, a microorganism belonging to the genus Serratia that is highly biotin-producing and transformed with a recombinant vector having a DNA encoding biotin synthetase of a microorganism belonging to the genus Bacillus,
and a method for producing biotin using the same.

Construction of a recombinant vector containing DNA encoding biotin synthetase derived from a microorganism belonging to the genus Bacillus

[0012] In the present invention, the microorganism used to collect the DNA encoding biotin synthetase may be any member of the genus Bacillus that has the ability to produce biotin, preferably a biotin-producing bacterium belonging to the genus Bacillus sphaericus. These microorganisms may be newly isolated from nature or known as long as they have DNA encoding biotin synthetase, and these microorganisms may be subjected to conventional microbial mutagenesis methods (for example, It may be obtained by a physical treatment such as ultraviolet rays, X-rays, or γ-ray irradiation, or a chemical treatment using a drug such as nitrosoguanidine. moreover,
In order to increase biotin production ability, these microorganisms may be given resistance to actitiazine acid or 5-(2-thienyl)valeric acid, either alone or both. Specific examples thereof include, for example, Bacillus sphaericus IF
O3525 and its N-methyl-N'-nitro-N
-Nitrosoguanidine-treated mutant strain (FERM P-6
422, FERM BP-1098, FERMP-6
143 etc. are exemplified.

[0013] There are no particular limitations on the method for collecting DNA encoding biotin synthetase from these microorganisms. For example, the total DN of a microorganism belonging to the genus Bacillus
A is isolated and purified by a conventional method, cut with a restriction enzyme, and then enzymatically ligated with a plasmid cut with a restriction enzyme that can produce the same sticky ends in DNA as the restriction enzyme, and the resulting recombinant plasmid is used. By transforming (or transducing) a biotin-requiring mutant strain that lacks the ability to biotin biosynthesize from desthiobiotin, and selecting from among the resulting transformants those endowed with the ability to produce biotin. , DN encoding biotin synthetase derived from Bacillus sp.
A transformed microorganism having A is obtained. Next, the recombinant plasmid integrated into the transformed microorganism is removed by a conventional method and treated with a restriction endonuclease to collect DNA encoding biotin synthetase derived from the genus Bacillus.

[0014] The DNA may also include a modified DNA having substantially the same function as the DNA, that is, a DNA in which a part of the base sequence has been substituted, inserted, or deleted. , those having the same functions are also naturally included.

[0015] Furthermore, as the above-mentioned DNA, one containing a Shine-Dalgarno sequence, a translation initiation codon, and a translation termination codon is preferably used.

[0016] When constructing a recombinant vector,
Plasmid vectors, λgt-based phage vectors, and the like are used.

[0017] As a vector for constructing a recombinant vector in the present invention, any vector can be used as long as it can transform (or transduce) a microorganism belonging to the genus Serratia, which will be described later. When using biotin-auxotrophic Escherichia coli as a host, use stringent-type pSC101, pRK353, pRK646, pR as EK-based plasmid vectors.
Relaxed ColE1 such as K248 and pDF41
, pVH51, pAC105, RSF2124, pCR
1, pMB9, pBR313, pBR322, pBR3
24, pBR325, pBR327, pBR328, p
KY2289, pKY2700, pKN80, pKC7
, pKB158, pMK2004, pACYC184,
λgt/λC, λgt/λB, λWES/λC, λ as λgt-based phage vectors such as pUC8 and pUC9.
WES・λB, λZJvir・λB', λALO・λB
, λWES・T5622, λDam, etc. are used. Among these vectors, plasmids are particularly preferred.

[0018] Furthermore, the DNA encoding biotin synthetase contained in the recombinant vector is a DNA having a promoter sequence that functions in the above-mentioned transformed strain.
(hereinafter simply referred to as a promoter) and must be incorporated downstream thereof. If there is no promoter upstream of the integration site of the DNA in the recombinant vector, place the promoter upstream of the DNA.
Just install it so that NA is controlled.

The promoter that can be used in the present invention may be either natural or artificially synthesized, as long as the recombinant vector incorporating the promoter can function in a microorganism belonging to the genus Serratia. Such promoters include those derived from Gram-negative bacteria, such as the lac promoter, lpp promoter, PL promoter, PR promoter, and Trp promoter derived from Escherichia coli.
Examples include a promoter, a Tac promoter, and a catechol-2,3-oxygenase promoter derived from a microorganism of the genus Pseudomonas. As for natural products, those contained in the vector used as a recombinant vector may be used as is.

DN encoding biotin synthetase
The method for preparing and isolating a recombinant vector incorporating A is that after the vector is introduced into a cell of a microorganism belonging to the genus Serratia, biotin derived from a microorganism belonging to the genus Bacillus is transferred into the cell.
There are no particular limitations as long as the synthetase can be prepared and isolated in such a way that it can be expressed. For example, methods for preparing recombinant plasmids may include restriction endonucleases (e.g. Ec
oRI, HindIII, BamHI, SalI, etc.) and then treated with DNA ligase (e.g., T4 DNA ligase, Escherichia coli DNA ligase, etc.), or depending on the cut end, terminal transferase, DNA polymerase, etc. Conventional methods such as treating with biotin synthetase and then using DNA ligase to join the DNA encoding biotin synthetase (Methods in Enzymology, 68, 41 (197
9); Genetic Manipulation Experimental Methods, p. 135 (Yasutaka Takagi, Kodansha, 1980).

DN encoding biotin synthetase
A method for selecting a recombinant vector having A is performed, for example, by the following procedure.

First, using a recombinant plasmid containing DNA encoding biotin synthetase, a mutant strain of Escherichia coli that is deficient in restriction endonucleases and requires biotin, such as Escherichia coli C6, is generated.
00 [Proceedings of National Academic Science in USA, 71, 4597 (1
974)], Journal of Bacteriology,
94, 1930 (1967), microorganisms lacking the ability to biosynthesize desthiobiotin into biotin, such as Escherichia coli K3 (pSB01) FERM-1099 (Japanese Patent Publication No. 62
-44889) to transform Escherichia coli K3 etc. obtained by curing. The resulting transformed cells were then transformed into the above-mentioned biotin-requiring Escherichia.
After the mutant strain of E. coli is spread on an agar plate medium in which biotin has been removed from a medium in which the mutant strain of E. coli can grow, it is cultured at 27 to 37° C. for 2 to 4 days, and the resulting colonies are isolated by fishing. At this time, the transformation method is not particularly limited, and for example, a method of increasing the membrane permeability of the host cell by treating the cells with a calcium chloride solution at low temperature and incorporating the recombinant plasmid DNA into the host [Journal of・Molecular Biology, 53, 159 (1970), etc.]. Next, plasmid DNA was extracted from some cells of the obtained transformed strain by a rapid method [Molecular Cloning, p. 365 [edited by Maniatis et al., Cold Spring Harbor Laboratory, (1982)]. The biotin-requiring Escherichia
After confirming that the microorganism obtained by transforming the cells of a mutant strain of E. coli does not require biotin, the remaining transformed strain is transformed using the cleared lysate method [Genetic Manipulation Experimental Methods, p. 125 (written by Yasutaka Takagi). A recombinant plasmid can be isolated by extracting the plasmid using a method such as 1980). Preferred examples of such recombinant plasmids include, for example, pSB301.
(Unexamined Japanese Patent Publication No. 63-44889).

DN encoding biotin synthetase
Construction of a transformed microorganism belonging to the genus Serratia using a vector having A The microorganism used for transformation in the present invention is a microorganism belonging to the genus Serratia, preferably a microorganism belonging to the genus Serratia. These microorganisms are
It may be a known strain isolated from a microbial strain repository or a strain newly isolated from the natural world.
For example, Serratia marcescens IFO12648 (a microorganism preserved at the Fermentation Research Institute), Serratia marcescens Sr41 (FERM BP-48)
7) etc. Furthermore, these microorganisms may or may not have the ability to produce biotin.

Microorganisms other than those belonging to the genus Serratia,
For example, with microorganisms belonging to the genus Enterobacter or Citrobacter, transformed microorganisms with high biotin-producing ability cannot be obtained.

DN encoding biotin synthetase collected from a Bacillus microorganism capable of producing biotin
The method for transforming a microorganism belonging to the genus Serratia with a recombinant vector having A is not particularly limited as long as the recombinant vector is introduced into the microorganism belonging to the genus Serratia and expressed. For example, a method of introducing a recombinant plasmid by treating bacterial cells with calcium chloride (e.g., Eric et al., Journal of General Microbiology, 133, 2053-2057, 198
7) Laws etc. Specific examples include the following methods.

First, a recombinant plasmid containing a DNA encoding biotin synthetase is isolated from, for example, Serratia sp.
Incorporated into cells of a wild strain of S. marcescens (for example, Serratia marcescens IFO12648 strain) by the method of Reid et al. [Gene, 17, 107 (1982)],
A transformed strain is obtained by isolating drug-resistant colonies. In this manner, it is possible to obtain a recombinant microorganism obtained by transforming a microorganism belonging to the genus Serratia with a recombinant plasmid having a DNA encoding biotin synthetase collected from a microorganism of the genus Bacillus having the ability to produce biotin.

[0027] In this way, it is possible to obtain a microorganism of the present invention, that is, a microorganism belonging to the genus Serratia that has been transformed with a recombinant vector having a DNA encoding biotin synthetase.

Production of biotin using a transformed microorganism belonging to the genus Serratia Biotin can be produced by culturing the above-mentioned transformed microorganism belonging to the genus Serratia by the following method.

The biotin production medium used in the present invention contains 10 to 20% of sugars such as glucose, sucrose, and molasses, organic acids such as fumaric acid and citric acid, or alcohols such as glycerol as carbon sources. , a medium containing 1 to 2% ammonium acetate or urea as a nitrogen source, and appropriate amounts of corn steep liquor, peptone, yeast extract, casein hydrolyzate, etc. as organic nutrients in the range of 0 to 1%. can be suitably used. In addition to these, add a small amount of potassium phosphate, magnesium sulfate, ferrous sulfate, sodium molybdate, etc., and further add calcium carbonate to maintain the pH of the medium at 6 to 8, or ammonia, etc. as necessary. It's okay. In the present invention, desthiobiotin can be added to the medium as necessary. The amount of desthiobiotin added is not particularly limited and may be appropriately selected depending on the biotin-producing ability of the microorganism.

[0030] The microorganism of the present invention is inoculated into the above medium, and 2
into the medium by culturing at 0 to 37°C, preferably 20 to 35°C, more preferably 23 to 28°C, under aerobic conditions such as shaking or aeration for 2 days or more, preferably 3 days or more. It can accumulate a significant amount of biotin. Sometimes, incubation for too long can reduce the amount of biotin in the medium. The produced biotin can be purified, for example, by removing bacterial cells and other insoluble materials after completion of the culture, and then by a known method, for example, the method described in Japanese Patent Publication No. 8918/1983. That is, biotin in a solution from which bacterial cells have been removed may be adsorbed on activated carbon, eluted with a solvent such as ammonia-containing ethanol, concentrated, and then applied to an anion exchange resin.

[0031]

[Examples] The present invention will be explained in more detail with reference to Examples below. In addition, in the examples, biotin was quantified using Lactobacillus plantarum.
plantarum) [Vitaminology Experimental Methods, Volume II, p. 486 [edited by the Japan Vitamin Society, Tokyo Kagaku Dojin (1985)]].

(Example 1) (1) Plasmid shedding Escherichia coli K3 (pSB01) FERM BP-
In order to eliminate pSB01 from 1099, this strain was inoculated into LB medium containing 10 μg/ml of acridine orange and cultured with shaking at 37° C. for 15 hours. After culturing,
100 μl of a 105-fold diluted culture solution was spread on an LB plate agar medium and cultured at 37° C. for 24 hours. After 24 hours of culture, the colonies that appeared were treated with 50 mg of ampicillin.
Replica onto LB plate agar medium containing /l and incubate at 37℃ for 15 minutes.
After culturing for several hours, strains that did not grow were selected. After culturing for 15 hours, the strain Scherichia coli K3-1, which grows on LB plate agar medium but not on LB plate agar medium containing ampicillin, was selected.

(2) Recovery of pSB01 Birunboim and Doly using Escherichia coli K3 (pSB01)
Method [Nucleic Acid Research (Nucle)
ic Acid Research), 7, 151
3 (1979)] with a total length of approximately 12.5 Kbp.
was recovered. Plasmid pSB01 is a DN encoding biotin synthetase of a microorganism belonging to the genus Bacillus.
This is a plasmid containing A. The restriction endonuclease cleavage map is shown in FIG.

(Example 2) Construction and analysis of hybrid plasmid pSB03 (1) Construction of hybrid plasmid 1μ of pSB01 plasmid DNA obtained in Example 1 (2)
completely digested with restriction endonuclease EcoRI, and 1 μg of the resulting mixture of three types of DNA fragments was diluted with 10 mM
Religion was performed by mixing with 3 units of T4 DNA ligase in 50mM Tris-HCl containing MgCl2, 1mM ATP and 10mM dethiothreitol pH 7.4 and reacting overnight at 4°C.

(2) Transformation of biotin-requiring E. coli and selection of transformed E. coli having biotin-producing ability Example 1
100m of Escherichia coli K3-1 obtained in (1) of
The cells were cultured with shaking for 2 and a half hours at 37° C. in 1 LB medium, and the cells were collected by centrifugation during the logarithmic growth phase. This bacterial cell was prepared using Kushner's method [Genetic Engineering].
g) p. 17, 1978].

Next, 0.1 μg of the T4 DNA ligase reaction mixture obtained in (1) was added to 0.2 m of the above permissible cell suspension.
1 and statically cultured at 0°C for 30 minutes. After keeping at 43.5°C for 30 seconds, add 1.8ml of LB medium and heat to 37°C.
The cells were cultured for 1 hour. This was centrifuged to collect bacteria. After washing the bacterial cells twice with 0.85% NaCl aqueous solution,
．． It was suspended in 1 ml of 85% NaCl aqueous solution. Next, 0.1 ml of the bacterial cell suspension was added to C containing 50 μg/ml ampicillin.
A medium [glycerin 2%, casamino acids (vitamin free) 1%, K2HPO4 0.2%, KH2PO4
0.1%, MgSO4.7H2O 0.005%, F
eSO4・7H2O 0.001%, MnSO4・4
~6H2O 0.001%, Thiamine Hydrochloride 0.00
001%] on a 1.5% agar medium and cultured at 37°C for two nights. The resulting colonies were ampicillin resistant and non-biotin auxotrophic, thus obtaining a transformed strain using a hybrid plasmid incorporating DNA carrying genetic information involved in biosynthesis of biotin. $ (3) Analysis of hybrid plasmid pSB03 The transformant obtained in Example 2 (2) was cultured with shaking overnight in 5 ml of CA medium containing 50 μg/ml ampicillin, and the cells were collected by centrifugation. method [Nucleic Acid Research (Nucleic Acid Research)
dRes. ) 7, 1513-(1979)], hybrid plasmid DNA was extracted. The smallest of the extracted plasmid DNAs has a total chain length of approximately 8.8k.
b, approximately 3.7k from pSB01 plasmid DNA
The EcoRI cleavage fragment of b was missing. This hybrid plasmid was named pSB03, and its restriction endonuclease cleavage map is shown in FIG.

(Example 3) Construction and analysis of hybrid plasmid pSB103 3 μg of pSB03 plasmid DNA obtained in Example 2 was completely digested with restriction endonuclease HindIII. Separately, 1 μg of pUC9 was completely digested with restriction endonuclease HindIII. Then pSB
A mixture of three types of DNA fragments obtained by cutting 03 (1
μg) and cleaved pUC9 (1 μg) at 10 mM
A reaction mixture was prepared by mixing with 3 units of T4 DNA ligase in 50mM Tris-HCl, pH 7.4, containing MgCl2, 1mM ATP and 10mM dethiothreitol. A hybrid plasmid was obtained by reacting each of these overnight at 4°C.

(2) Transformation of biotin-auxotrophic Escherichia coli A transformed strain using the hybrid plasmid was obtained in the same manner as in (2) of Example 2, except that 1 μg of the reaction mixture in (1) of Example 3 was used. .

(3) Hybrid plasmid pSB10
Analysis of 3 The transformed strain obtained in (2) of Example 3 was added at 50 μg/m
The cells were cultured with shaking overnight in 5 ml of CA medium containing ampicillin and collected by centrifugation, and then hybrid plasmid DNA was extracted by the method of Birnboim and Dawley described above. The smallest of the extracted plasmid DNAs had a total chain length of about 6.2 kb. This plasmid is pSB03
An approximately 2.0 kb HindIII cleavage fragment of plasmid DNA was inserted into the pSB103, and it was named pSB103.

[0040] The DNA fragment derived from Bacillus sphaericus IFO03525 contained in this inserted fragment is MboI
It is approximately 1.5 kb from HindIII to HindIII, and its detailed restriction endonuclease cleavage map is shown in FIG.

(Example 4) Construction of hybrid plasmid pSB301 (see FIG. 4) The following operation was performed to directly link DNA encoding biotin synthetase to the promoter operon region of the Trp operon. After completely digesting pSB103 with restriction endonuclease MaeI, the digested DNA fragment (2.0 μg) was added to 30 mM sodium acetate (pH 4.
5), 250mM NaCl, 1mM ZnSO4, 5
% glycerol with 1 unit of S1 nuclease;
Both ends of the DNA fragment were made blunt by reacting at 37°C for 30 minutes. The reaction product was then subjected to agarose gel electrophoresis, and a DNA fragment of approximately 2.1 kb was recovered.

On the other hand, pDR720 (manufactured by Pharmacia), which contains the promoter operator of the Trp operon and has ampicillin resistance as a marker, was completely digested with restriction endonuclease SmaI and then treated with alkaline phosphatase.

Approximately 2.1 kb recovered from agarose gel
DNA fragment (0.1 μg) and cleaved pDR720
(0.1μg) in 10mM MgCl2, 1mMAT
pH 7.4 containing P and 10mM dethiothreitol
A reaction mixture containing the hybrid plasmid was obtained by mixing with 2 units of T4 DNA ligase in 50 mM Tris-HCl and reacting at 15° C. for 4 hours.

A transformed strain using the hybrid plasmid was obtained in the same manner as in Example 2 (2) except that 0.2 μg of this reaction mixture was used.

The obtained transformant was cultured overnight in 3 ml of LB medium containing 50 μg/ml ampicillin, and after harvesting by centrifugation, hybrid plasmid DNA was extracted by the method of Birnboim and Doory. The smallest of the extracted plasmid DNAs had a total chain length of about 6.1 kb. In this plasmid, the translation initiation codon GTG of the biotin synthase gene derived from Bacillus sphaericus began approximately 70 bases downstream of the transcription initiation site of the Trp promoter. This was named pSB301.

(Example 5) Obtaining a transformed strain using Serratia marcescens IFO12648 as a host Using pSB301 obtained in Example 4, transfection was carried out by the method of Reid et al. [Gene, 17, 107 (1982)]. That is, Serratia marcescens IFO12648 was grown in LB medium (Bactotryptone 1%, yeast extract 0.5%, Na
Cl1%; adjusted to pH 7.5), cultured in 20 ml at 30°C with aeration and shaking, and when the bacterial cell concentration reached 2 x 108 cells per ml, the bacterial cells were incubated at 12,000 x g for 10 minutes. Collected by centrifugation. 10ml of this bacterial body
M, suspended in cold NaCl solution. This suspension was heat-treated at 65°C for 1 minute, cooled on ice, collected by centrifugation, the supernatant was discarded, and then heated in 10ml of 30mM cold Ca at 0°C.
After suspension in l2 solution and cooling, 3000 x g at 5°C.
, collect bacteria by centrifugation for 5 minutes, discard the supernatant, and then
1 of cold 30mM CaCl2, 15% glycerol solution. Add 0.1 μg of plasmid to 0.2 ml of this CaCl2 treatment solution and heat at 42°C for 75 seconds (approximately 2 to
5 x 108 cells/ml) after heat treatment, 0 for 1 to 24 hours.
After keeping at ℃, ampicillin 500μg/ml L
The transformed strain Serratia marcescens IFO12648 (pSB301) was obtained by plating the B plate medium and culturing it for two nights at 28°C, and then separating the grown colonies.
get. It was confirmed that the plasmid DNA contained in this transformed strain was pSB301 by using the restriction endonuclease cleavage site.

(Example 6) Biotin production example 1
Serratia marcescens IFO12648 obtained from
(pSB301) and Serratia marcescens IFO12648 as a control strain were each pre-cultured on LB plate medium overnight. The fermentation medium described in Japanese Patent Publication No. 2-27980 to which 100 mg/l of ampicillin was added (sucrose
0%, urea 1.0%, K2HPO4 0.1%, Mg
SO4・7H2O 0.1%, FeSO4・7H2O
0.01%, CaCO3 1%) and DTB as a precursor added to a final concentration of 1 mg/ml (pH 7.0). ) Inject into test tubes and inoculate one platinum loop of each of the above-mentioned bacterial cells. Then culture is carried out at 25°C with reciprocal shaking. The amount of biotin produced after 96 hours is shown in Table 1.

[0048]

[Table 1]

(Comparative Example 1) Citrobacter freundii
IFO13544 (pSB301), in which plasmid pSB301 was introduced into IFO13544, Enterobacter cloacae (Enterobacter cl.
oacea) IFO12935 with plasmid pSB30
IFO12395 (pSB301), which introduced 1
Enterobacter aerogenes
ter aerogenes) IFO12010 in which plasmid pSB301 was introduced into IFO12010
(pSB301) by the method described in Example 2.
Biotin was produced from TB. On the other hand, Escherichia
Escherichia coli MC169 (pSB301), in which plasmid pSB301 was introduced into E. coli MC169, was
0 μg/ml ampicillin and 500 μg/ml DT
PC medium containing B (2% glycerin, 5% proteose peptone, 2% casamino acids, 1% K2HPO4, K
Cl 0.05%, MgSO4・7H2O 0.0
5%, MnSO4.4~6H2O 0.001%, F
The cells were inoculated into 3 ml of a 1.2 times concentrated medium (eSO4.7H2O 0.001%, pH 7.0), and after shaking culture at 37°C for 1 hour, indole acrylic acid was added at 16.7 μg/ml to induce the Trp promoter. ml, and cultured with shaking at 37° C. overnight. The amount of biotin produced by each strain is shown in Table 2.

[0050]

[Table 2]

[0051]

Thus, according to the present invention, production of biotin can be achieved by transforming a microorganism belonging to the genus Serratia with a recombinant plasmid having a DNA encoding biotin synthetase derived from a microorganism of the genus Bacillus that has the ability to produce biotin. Microorganisms with excellent performance can be obtained, and biotin can be efficiently produced by culturing these microorganisms.

[0052]

[Brief explanation of the drawing]

FIG. 1 is a restriction endonuclease cleavage map of pSB01 plasmid DNA.

FIG. 2 is a restriction endonuclease cleavage map of pSB03 plasmid DNA.

FIG. 3 is a restriction endonuclease cleavage map of biotin synthetase DNA derived from a microorganism belonging to the genus Bacillus.

FIG. 4 shows a method for preparing pSB301 plasmid DNA.

Claims (2)

[Claims] 1. A microorganism belonging to the genus Serratia that has been integrated with a recombinant vector having a DNA encoding an enzyme involved in the reaction of converting desthiobiotin into biotin in a microorganism belonging to the genus Bacillus. 2. A method for producing biotin, which comprises culturing the microorganism according to claim 1 under aerobic conditions, and then recovering biotin from the culture medium. 0001