JPH0227980A - Novel microorganism and preparation of d-biotin with the same microorganism - Google Patents

Abstract

PURPOSE:To profitably prepare d-biotin by forming by a biological engineering method an microorganism belonging to the genus Serratia, having resistance against biotin structure-like substances and having an ability to produce and store the d-biotin. CONSTITUTION:A deoxyribonucleic acid collected from microorganisms belonging to the genus Serratia having an ability to produce and store d-biotin and governing the production and storage of the d-biotin is recombined into a vector.plasmid and the recombined plasmid is inserted into a microorganism belonging to the genus Serratia. The microorganism belonging to the genus Serratia having the d-biotin-producing and storing ability is cultured in a medium to produce and store the d-biotin in the medium, followed by collecting the produced and stored d-biotin. The microorganism includes Serratia.marcescens TA5021, TA5022 and TA5023 (FERM No. 10118), etc.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a novel microorganism and a method for producing d-biotin using the same.

(Prior Art) d-Biotin is an essential vitamin for humans and animals, and is important as a pharmaceutical raw material or feed additive.

Conventionally, using a normal fermentation medium containing carbon and nitrogen sources,
As a method for producing d-biotin by fermentation, a method using microorganisms of the genus Streptomyces and Micromonospora (Japanese Patent Publication No. 41-21756) is known.
Furthermore, as a method using genetic recombination technology, there is a method using microorganisms of the genus Nijierisia (Japanese Patent Application Laid-Open No. 61-2026
86, 62-155081) is known.

(Structure and Effects of the Invention) As a result of intensive research, the present inventors discovered that, quite unexpectedly, microorganisms of the genus Serratia were mutated to produce actitiazine acid and
-(2-thienyl)-n-valeric acid and other substances with a biotin structure similar to those that confer resistance, and that the ability to produce d-biotin is significantly enhanced, and that this mutant strain produces and accumulates d-biotin. Chromosome deoxyribonucleic acid (
After cutting out a fragment (hereinafter abbreviated as DNA) and incorporating it into a vector plasmid, the resulting recombinant plasmid is transferred to a microorganism of the genus Serratia that has the ability to produce and accumulate d-biotin. The present inventors have discovered that the production capacity can be significantly increased and that d-biotin can be advantageously produced industrially by using the microorganism, and have completed the present invention.

That is, the present invention based on such knowledge provides: (i) a microorganism that belongs to the genus Serratia, has resistance to biotin structurally similar substances, and has the ability to produce and accumulate d-biotin; (ii) a microorganism that has the ability to produce and accumulate d-biotin. A microorganism in which a microorganism belonging to the genus Serratia contains a recombinant plasmid in which a deoxyliponucleic acid responsible for producing and accumulating d-biotin, which is collected from a microorganism belonging to the genus Serratia, is incorporated into a vector plasmid; (iii) a microorganism having the ability to produce and accumulate d-biotin; By culturing a microorganism belonging to the genus in a medium, d-biotin is produced and accumulated in the medium.

A method for producing d-biotin, which comprises collecting the d-biotin. It is.

Microorganisms belonging to the genus Serratia (Verse's Manual of Systematic Bacteriology, Vol. 1, p. 477, 1984), especially microorganisms belonging to Serratia marcescens, are used as parent strains for preparing the mutant strains of the present invention. Ru. The strain may be a known strain distributed from a microbial strain preservation institution or a strain newly isolated from nature, such as Serratia marcescens 5r41 (Feikoken Jokyo No. 487). can give. Furthermore, mutant strains such as various amino acid auxotrophic strains, various nucleic acid auxotrophic strains, or various vitamin auxotrophic strains other than biotin auxotrophic strains may be derived from wild-type strains by mutation.
A specific example of such a strain is Serratia marcescens 5r41 isoleucine auxotroph strain D-60 (Journal of General Microbiology, "■,
51 (1980)).

By imparting resistance to biotin structurally similar compounds to such a parent strain, a mutant strain with a high ability to produce and accumulate d-biotin can be obtained. For example, when using the wild strain Serratia marcescens 5r41, the cells of the strain can be After treatment with a mutagenic procedure, biotin structural analogues such as actitiazine acid (hereinafter abbreviated as ACM), 5-(2-thienyl)-n-valeric acid, and α-dehydrobiotin were added at 0.1 to 1 O. /- containing minimal agar plate medium, such as Davis and Mindioli's minimal medium [
Journal of Bacteriology, 60.1'7
(1950)] or in a culture medium in which the carbon source has been changed to various type I, organic acids, or amino acids at 27 to 37°C.
After culturing for 7 days, a strain resistant to biotin structural analogs is obtained by separating the resulting large colonies. Next, the d-biotin secretion ability of these resistant strains was determined according to the halo method [Journal of Bacteriology 79, 754 (1960)] using d-biotin-requiring mutant strains of the genus Serratia or genus Elysia as indicator bacteria. A strain that forms a halo, that is, a strain that has an increased ability to produce and accumulate d-biotin, is selected. More specifically, cells of Serratia marcescens 5r41 were prepared using the method of Ablebarb et al. [Biochemical and Biophysical Research Communications, 18.788 (1)].
965)) gives N-methyl-N゛-nitro-N-
After treatment with 100 μg/- of nitrosoguanidine for 30 minutes, a-ACMo, a minimal agar plate medium containing 5 μg/lli of ammonium succinate, 0.1% of ammonium sulfate, 0.7% of dipotassium phosphate, Potassium phosphate 0.3%, magnesium sulfate heptahydrate 0.01%,
Apply on agar (1.5%). Next, ACM-resistant strains can be obtained by culturing at 30° C. for 4 days and separating the resulting large colonies. By selecting a strain capable of secreting d-biotin from among such resistant strains using the halo method using a d-biotin-requiring strain as an indicator tooth, Serratia spp.
A mutant strain of S. marcescens having the ability to produce and accumulate d-biotin can be obtained. Also, ACMo, 5■/
Cells of a mutant strain that is resistant to Ml and has the ability to produce and accumulate d-biotin are subjected to mutagenesis treatment in the same manner as above, for example, 2■/IRI.
After coating on ACM-containing minimal agar plate medium and performing the same operation, the resulting ACM-resistant strain is subjected to the halo method, and strains that form a halo significantly larger than the parent strain are selected to produce d-biotin. A with increased storage capacity
CM-resistant strains can be obtained. In addition, when isolating a strain resistant to a biotin structural analogue, a plurality of structural analogue substances, such as ACM and 5-(2-thienyl)-〇-
Minimal agar plates supplemented with both valeric acid and valeric acid can also be used.

Examples of mutant strains obtained as described above include Serratia marcescens 5B prepared in Example 1 of the present invention.
303 (M Koken Bacteria No. 10119) or Serratia
Marcesoscens 5B411 is mentioned.

(2) Preparation of a recombinant plasmid that stores d-biotinylation ■ Preparation of chromosomal DNA In the present invention, the chromosomal DNA that controls d-biotin biosynthesis
Among enzymes involved in biotin synthesis in microorganisms, enzymes that catalyze the synthesis of bimeloyl-coenzyme A, 7-kedo-8-aminopelargonic acid synthase, 7,8-diaminopelargonic acid aminotransferase, dethiobiotin synthetase, and Contains all or part of the DNA that carries the genetic information for biotin synthetase. The above enzyme is -1 to N. coli
In 2, bio Cs bio F% bio
A% bi.

These enzymes correspond to enzymes defined by five genes named d- and bio-B.
The chromosomal DNA that controls d-biotin synthesis used in the present invention is subject to strong feedback inhibition by biotin.
It is desirable that the chromosomal DNA is a mutant type that is released from feedback inhibition.The donor microorganism for such chromosomal DNA may be any strain as long as it belongs to the genus Serratia and has the ability to produce d-biotin.For example, the above-mentioned Serratia・Serratia marcescens 5r41 and mutant strains with released feedback inhibition, Serratia marcescens, fuscens 5
B303 and Serratia marcescens 5R411. To collect chromosomal DNA from these microorganisms, for example, microbial cells are treated with lysozyme, treated with a surfactant (sodium lauryl sulfate, sarcosyl (sodium N-lauroyl sarcosinate), etc.), deproteinized, and treated with ethanol. Conventional method for precipitation [Journal of Molecular Biology, 3.20B (19
61) ; Biochimica Et Biophysica Acta, 72.619 (1963)).

The chromosomal DNA obtained as described above may be used as it is, or after removing unnecessary parts for biotin synthesis, it may be converted into recombinant DNA.
It can be used to prepare A.

■ Preparation of vector/plasmid DNA The vector/plasmid that incorporates the chromosomal DNA as described above is not particularly limited as long as it can be transferred into cells of microorganisms belonging to the genus Serratia and can be replicated in the transferred cells, but examples include pSCIOI ( Proceedings of the National Academy of Sciences USA, 70.3240 (1973)
), pLG339 (Gene, ■, 332 (19
82)), pBR322 (Gene, 1, 95 (19
77) ), pBR325 [Gene, 4.121
(197B)), pAcYc1?7 (Journal of Batatteriology, U(, 1141 (1978
)) and pKPT1124 (Journal of Biotechnology, 3.47 (1985)), various vector plasmids with different copy numbers, promoters and/or drug resistance markers can be used. The above plasmids were obtained using the cleared lysate method [Gene Manipulation Experimental Method 1] from N. coli cells that carry them.
Page 25 (Yasutaka Takagi, Kodansha, 1980); Molecular Cloning, page 86 (edited by Maniais et al.,
Cold Spring Harbor Laboratory 119
It can be prepared by a conventional method such as [1982]].

■ Preparation of recombinant plasmid DNA Chromosomal DNA and vector plasmid D obtained above
Preparation of recombinant plasmids from NA and restriction endonucleases (e.g. II! coRI% old ndDI, B
chromosomal DNA using amHI, Sal r, etc.)
After cutting the ti and plasmid DNA strand, it is treated with DNA ligase (for example, T4 DNA ligase or Escherichia coli DNA ligase, etc.), or depending on the cut end, it is treated with terminal transferase, DNA polymerase, etc., and then DNA ligase is applied. Conventional methods such as joining DNA strands [Methods in Enzymology,
41 (1979): Gene Manipulation Experimental Methods, p. 135 (Yasutaka Takagi, Kodansha, 1980)].

■ Selection of a recombinant plasmid capable of producing and accumulating d-biotin and preparation of its DNA Selection of a recombinant plasmid capable of producing and accumulating d-biotin is carried out using the recombinant plasmid DNA obtained in step (■) above, using restriction endonuclease Mutant strains of N. coli that are deficient and require d-biotin [e.g., N. coli strain 3911776 (ATCC 31244, Molecular Cloning Op Recombinant DNA
, 248 pages, Scott and Wehner, eds.
Press, 1977), and then the resulting transformed cells were applied to an agar plate medium in which d-biotin had been removed from the medium in which the above-mentioned N. coli mutant strain could grow. This can be carried out by culturing at 27 to 37°C for 2 to 4 days and separating the resulting colonies. At this time, transformation is carried out by, for example, treating cells with a calcium chloride solution at low temperatures to increase the membrane permeability of the host cells, and incorporating the recombinant plasmid DNA into the host cells [Journal of Molecular Biology]. 159 (1970)).

Preparation of recombinant plasmid DNA from the cells of the transformant obtained as described above can be carried out, for example, using the rapid method [Molecular Cloning, 3651 (edited by Maniathes et al., Cold Spring Harbor Laboratory)]. 11982)]
After confirming that the strain obtained by transforming cells of a mutant strain of N. coli that requires d-biotin does not require d-biotin, a plasmid was extracted from the transformed strain by the cleared lysate method. This can be carried out by extracting DNA. In this way, a recombinant plasmid DNA is obtained in which a chromosomal DNA fragment of Serratia marcescens having the ability to produce and accumulate d-biotin is inserted into a vector plasmid DNA.

The microorganism of the present invention, that is, a microorganism in which a microorganism belonging to the genus Serratia is made to contain a recombinant plasmid incorporating a chromosomal DNA fragment controlling the ability to produce and accumulate d-biotin collected from a microorganism belonging to the genus Serratia that has the ability to produce and accumulate d-biotin,
For example, it can be prepared as follows.

First, the recombinant plasmid DNA having the ability to produce and accumulate d-biotin obtained in (2) above is transferred to a restriction endonuclease-deficient strain of Serratia marcescens (for example, Serratia marcescens 5r41 strain 77392 [Journal of Bacteriology, 161.1 (
1985) ) etc.) using Takagi and Kisumi's method [Journal of Bacteriology, Dan 1, l.
(1985)) to obtain a transformed strain. After culturing this transformed strain, plasmid DNA is isolated and purified from the resulting cells by the cleared lysate method. The thus obtained recombinant plasmid DNA having the ability to produce and accumulate d-biotin has been modified by the Serratia marcescens modification system. Next, the above recombinant plasmid DNA is introduced into cells of the host strain by the Takagi and Kisumi method, and drug-resistant colonies are isolated to obtain a transformed strain. This host strain may be a wild strain or a mutant strain as long as it belongs to the genus Serratia, but strains with a higher ability to produce and accumulate d-biotin are particularly preferably used.

In this manner, the d-
A microorganism belonging to the genus Serratia can be obtained by containing a recombinant plasmid incorporating a chromosomal DNA fragment responsible for the ability to produce and accumulate biotin. Such microorganisms include, for example, Serratia marcescens TA5021.
, TA5022, TA5023 (Fiber Engineering Laboratory No. 101
No. 18) and TA5024.

Among the above recombinant strains, TA5021 and TA5022 are microorganisms in which Serratia marcescens 5r41 contains a recombinant plasmid having the ability to produce and accumulate d-biotin using pBR322 or pLG339 as a vector, and TA5023 and TA5024 are Serratia marcescens 5r41 microorganisms. pLG in each of the 5B2O3 and 5B411 strains.
These microorganisms contain a recombinant plasmid having the ability to produce and accumulate d-biotin using 339 as a vector, and all strains have morphological characteristics of Serratia marcescens 5r41.

(4) Production of d-biotin d-biotin can be produced by culturing the microorganism thus obtained in a medium and collecting the d-biotin produced and accumulated in the medium.

The d-biotin production medium used in the present invention contains 10 to 20% of a carbon source such as Class Ii such as glucose, sucrose or molasses, an organic acid such as fumaric acid or citric acid, or an alcohol such as glycerol. 1 to 2% of organic ammonium salts such as ammonium acetate, inorganic ammonium salts such as ammonium sulfate and ammonium chloride, or urea as a nitrogen source, and 0% of corn steep liquor, peptone, yeast extract, casein hydrolyzate, etc. as organic nutrients. A medium containing an appropriate amount of each in the range of 1% to 1% can be suitably used. In addition to these, add a small amount of potassium phosphate, magnesium sulfate, ferrous sulfate, sodium molybdate, etc. 1, and further adjust the pH of the medium to 6 to 8.
In order to maintain the temperature, calcium carbonate or ammonia or the like may be added as necessary.

According to the present invention, the above-mentioned medium is inoculated with the microorganism of the present invention,
A significant amount of d-biotin can be accumulated in the medium by culturing at 25°C to 37°C under aerobic conditions such as shaking culture or aerobic agitation for 2 to 7 days.

Purification of the produced d-biotin can be carried out by removing bacterial cells and other insoluble matter after the completion of the culture, and then following a known method, for example, the method described in Japanese Patent Publication No. 8918/1983. That is, it can be carried out by adsorbing d-biotin in a solution from which bacterial cells have been removed onto activated carbon, eluting it with a solvent such as ammonia-containing ethanol, concentrating it, and then applying it to an anion exchange resin.

The present invention will be explained below with reference to Examples.
- Biotin determination method using Lactobacillus plantarum [Biochemistry Experiment Course, Vol. 13, p. 355 (
Edited by Yoshitsugu Nose et al., Tokyo Kagaku Dojin (1975)]. Additionally, dethiobiotin synthetase activity was determined using the method of Krell and Eisenbarb (The Journal of
Biological Chemistry, 6558 (1
970))).

Example 1 (Obtaining an ACM-resistant strain) (1) Cells of Torihiro Serratia marcescens 5r41 (It Koken Joyori No. 487), an ACM low-r#100 strain, were mutagenicly treated by the method of Ablebarb et al. and nutrient medium (glucose 0.5%, peptone 1.0%, meat extract 0.3%, yeast extract 1.0%,
Incubate for 1 hour in sodium chloride (0.5%). This culture solution is centrifuged, and the resulting cells are washed three times with physiological saline by centrifugation.

The cells were suspended in physiological saline, a-ACMO, 5 days/day.
- Minimal agar medium containing (0.5% ammonium fumarate)
, potassium phosphate 0.3%, dipotassium phosphate 0.7
%, magnesium sulfate heptahydrate 0.01%, agar 1.5
%), plate 1-10 x 10' cells per plate. After culturing at 30° C. for 3 to 5 days, ACM-resistant strains are obtained by separating the resulting large colonies.

Next, the d-biotin secretion ability of the ACM-resistant strain was examined by the halo method using the d-biotin-requiring strain of Serratia marcescens 5r41 as an indicator strain, and Serratia marcescens 5B3 was selected as one of the strains forming a large halo.
03 (Feikoken Bibori No. 10119) was obtained.

Furthermore, when the dethiobiotin synthetase activity of this strain was investigated, it was found that the parent strain Serratia marcescens 5r
This strain showed 10 times higher activity than d-
It was found that the feedback inhibition by biotin was released.

(2) ACMt? 1 to io x to' cells of Serratia marcefuscens 5B303 are plated on a minimum agar medium containing ff-ACM2.0■/- per plate, and cultured at 30°C for 5 to 7 days. A strain resistant to high concentrations of ACM is obtained by separating the resulting large colonies. This high concentration resistant strain was examined for d-biotin secretion using the same halo method as described in (1) above, and Serratia marcescens 5B411 was obtained as an ACM resistant strain that forms a significantly larger halo than the parent strain. .

This strain has about twice as much dethiobiotin as 5B303.
It showed synthetase activity.

Example 2 (Preparation of a recombinant plasmid having the ability to produce and accumulate d-biotin using pBR322 as a vector) (1) 0 amount of negative DNA - Serratia marcescens 5B obtained in Example 1-(1)
303 in L-broth (peptone 1%, yeast extract 0.5
%, sodium chloride 0.5%, pn 7.0) 1. O
1, cultured with shaking at 30°C for 4 hours, and the cells in the logarithmic growth phase were collected by centrifugation. After the bacterial cells are treated with lysozyme, they are lysed by treatment with sodium lauryl sulfate, and protein is removed by treatment with phenol. Next, chromosomal DNA was precipitated by treatment with ethanol, and this precipitate was treated with ribonucleic acid degrading enzyme (RNase, final concentration 10 μg/d).
was added and treated at 37°C for 1 hour to obtain purified chromosomal DNA 7°0.

(2) Vector plasmid pBR322 (ATCC3701
7) -12 C600 to N. coli holding
rf strain (ATCC33525) with L-pros 8001# containing 0.2% glucose! After culturing with shaking at 37°C for 166 hours, the bacterial cells were collected by centrifugation. After the resulting bacterial cells were lysed by treatment with lysozyme and sodium lauryl sulfate, sodium chloride was added to the final concentration of IM and centrifuged (100,000 ml).
0x g for 30 minutes). After collecting the supernatant and treating it with phenol, ethanol is added and centrifuged. The precipitate was dissolved in an aqueous solution of 10 sM) lithium hydrochloride-1++M disodium ethylenediaminetetraacetate (pH 7.5) and subjected to cesium chloride-ethidium bromide equilibrium density gradient centrifugation (
The plasmid DNA is separated and purified using 200,000 x g for 16 hours. In this way, 0.5 μ of DNA of vector plasmid pBR322 is obtained.

(3) 20 μg of the chromosomal DNA obtained in (1) above of the plasmid DNA is treated with restriction endonucleases Hindu and EcoRI simultaneously under normal conditions to partially cleave the DNA strand. Also, the above (
7 μg of the vector plasmid DNA obtained in 1) was
nd[l and EcoRI are simultaneously applied under normal conditions to completely cleave DNA13m. After heat treating both at 65° C. for 10 minutes, they are mixed and treated with T4 phage-derived DNA ligase under normal conditions to join the DNA strands to obtain recombinant plasmid DNA.

Nizielysiae, which is restriction endonuclease deficient and lacks the biotin operon (bioABPcD).
3911776 strains (^TCC31244) to χ1776
Nutrient medium for strains [2.5% Batatotributone, 0.75% yeast extract, 2 d of Tris-HCl (pH 7,5), 0.5 sM of magnesium chloride, MO1O of diaminopimeline
1%, thymidine 0.004%, glucose 0.5%] 1
5-, and cultured with shaking at 37°C. Bacterial cells that have grown to the middle of the logarithmic growth phase are collected by centrifugation.

0, 1? l Magnesium chloride solution 1
After suspending in 5-, collect the bacteria and suspend in ice-cold 0.1M calcium chloride solution 3-. Add this cell suspension to the above (
After adding the recombinant plasmid DNA obtained in step 3) and cooling with water for 30 minutes, the DNA is incorporated into the cells by heat treatment at 42° C. for 2 minutes. Next, nutrient medium 15w11 for χ1776 strain was added to this suspension, and the mixture was incubated at 37°C for 1 hour.
After incubating with shaking for an hour, collect the bacteria and add 15-30 ml of physiological saline for 30 min.
Wash twice. The bacterial cells were suspended in physiological saline 15-1, and this suspension 0.1-0.5- was mixed with χ17 containing 50 μg/- of ampicillin and 0.1 μg/- of dethiobiotin.
Minimal agar medium for 76 strains (glucose 0.5%, ammonium sulfate 0.1%, dipotassium phosphate 0.7%, potassium phosphate 0.3%, magnesium sulfate heptahydrate 0.0
1%, diaminopimelic acid 0.01%, thymidine 0.0
04%, casein hydrolyzate 0.2%, agar 1.5%)
and cultured at 37°C for 2 days. The resulting colonies were isolated, and transformed strains having the ability to secrete d-biotin were selected by the halo method. The bacterial strain thus obtained is χ1
The 776 strain nutrient medium 11 is inoculated, and the plasmid DNA is extracted and purified in the same manner as in (2) above to obtain 360 μg of recombinant plasmid pBM201 DNA. This recombinant plasmid DNA has a total chain length of about 11.3 kb, about 7
A DNA fragment of kb was inserted.

In addition, using the plasmid DNA, N. elissia 39
Cells of the 11776 strain were transformed and ampicillin-resistant re-transformed strain IO strain was selected on a nutrient agar medium for the χ1776 strain, and the growth of these strains was examined on a minimal agar medium for the χ1776 strain. showed growth when 7-kedo-8-aminopelargonic acid, 7,8-diaminopelargonic acid, or dethiobiotin was added, but did not show growth when they were not added.

Furthermore, all of these 10 strains showed the ability to secrete d-biotin. Therefore, this plasmid is a recombinant plasmid with the ability to produce and accumulate d-biotin, and its inserted DNA is thought to encode a gene group corresponding to the -12 biotin operon in N. coli. .

Example 3 (Preparation of a recombinant plasmid capable of producing and accumulating d-biotin using pLG339 as a vector) (1) Using the vector plasmid pLG339 (ATCC3713
1) Bacterial strain containing [Gene, ■, 335 (1
The vector plasmid pLG3 was prepared using the same method as described in Example 2-(2).
39 DNA 0°3■ is obtained.

(2) Example 2-(4) of plasmid DNA
2 μg of the DNA of the recombinant plasmid pBM201 having the ability to produce and accumulate d-biotin obtained in 2009 was treated with endonucleases EcoRI and HinduI simultaneously under normal conditions to completely degrade the DNA strand. Also above (
DNA of vector plasmid pLG339 obtained in 1)
1 μg was first completely decomposed using EcoRI under normal conditions, and then partially decomposed using ) A recombinant plasmid is obtained by ligating the DNA strands by allowing phage-derived DNA ligase to act under normal conditions.

(3) d-biotinylation using LG339 as a vector
Acquisition of accumulated biotin-producing bacterial strain) N. coli χ1776 strain was obtained from Example 2-(4).
After culturing and obtaining bacterial cells by the method described in Example 2.
- (4) Introducing the recombinant plasmid DNA obtained in (2) above into cells by the same method as described,
Example 2 - 0.1 to 0.5 of the cell suspension prepared in the same manner as in (4) was applied to a minimal agar medium for the χ1776 strain containing 100 μg/− of kanamycin sulfate and 0.2 μgod of d-dethiobiotin. , and culture at 37°C for 2 days. The resulting colony is isolated, and a transformation method having the ability to secrete d-biotin is obtained by the halo method. From the transformed strain, plasmid D was obtained according to the method described in Example 2-(2).
NA is extracted and purified to obtain 175 μg of DNA of recombinant plasmid pLGM201.

Example 4 (Implemented using Serratia marcescens d-extranuclease-deficient and restriction endonuclease-deficient Serratia marcescens 5r41 strain 77392, which carries a recombinant plasmid capable of producing and accumulating d-biotin, as a host. The DNA of the recombinant plasmid pBM201 obtained in Example 2-(4) and the DNA of the recombinant plasmid pLGM201 obtained in Example 3-(3) were introduced into cells of strain 77392 by the method of Takagi and Kisumi, and the respective traits Obtain transformants.Next, each transformant was inoculated into L-pros 1j! containing 0.2% glucose,
After culturing with shaking at 30°C for 16 hours, the bacteria were collected and used in Example 2-
98M201 and p modified by Serratia marcescens 5r41 by treatment similar to (2).
Obtain 250 μg and 150 μg of LGM201 DNA, respectively.

Using the DNA of 98M201 obtained in the above (1) and Serratia marcescens 5r41, a transformed strain Serratia marcescens TA5021 with ampicillin resistance is obtained by the method of Takagi and Kisumi. It was confirmed by a method using restriction endonuclease cleavage sites that the plasmid DNA contained in this transformed strain was the same as the 98M201 DNA obtained in Example 2-(4).

Further, using the DNA of ρLGM201 obtained in (1) above and Serratia marcescens 5r41, Serratia marcescens TA5022 with kanamycin resistance is obtained by the same method as above.

It was confirmed by a method using restriction endonuclease cleavage sites that the plasmid DNA contained in this transformed strain TA5022 was the same as pLGM201 DNA.

DNA of pLGM201 obtained in (1) above and Serratia marcescens 5B3 obtained in (1) of Example 1
03 and transformant strain Serratia marcesoscens TA5023 (
10118).

It was confirmed by a method using restriction endonuclease cleavage sites that the plasmid DNA contained in this transformed strain TA5023 was the same as pLGN201 DNA.

DNA of pLGM201 obtained in (1) above and Serratia marcescens 5B4 obtained in (2) of Example 1
11 strains to obtain a transformed strain Serratia marcescens TA5024 with kanamycin resistance in the same manner as in (2) above.

It was confirmed by a method using restriction endonuclease cleavage sites that the plasmid DNA contained in this transformed strain TA5024 was the same as pLGM201 DNA.

Example 5 (Production of d-biotin using an ACM-resistant strain of Serratia marcescens and a recombinant plasmid-carrying strain) 5B3 of Serratia marcescens obtained in Example 1
03 and 5B411 and Serratia marsenoscens TA5021, TA5022, TA50 obtained in Example 4
23 and TA5024 and Serratia marcescens 5R41 as a control strain were each treated with 500 μg of ampicillin.
or L containing 100 μg/IR1 of kanamycin sulfate
~broth, or those containing none of them After overnight culture in broth slant medium, fermentation medium [Yt 110%, urea 1.0%, dipotassium phosphate 0.1%, magnesium sulfate heptahydrate 0.1 %, ferrous sulfate heptahydrate o, o i%
and 15 ml of a solution containing 1% calcium carbonate was poured into a 500-capacity shaking flask and sterilized (however, the seal
Sugar was added separately after heat sterilization. pH 7, 0)], respectively. Then, culture is performed at 30° C. with reciprocating shaking (amplitude: am, 120 revolutions/min).

The amount of d-biotin produced and accumulated in the medium after 96 hours is shown in Table 1 below.

Table 1 Example 6 (Production of d-biotin in jar fermentor) Serratia marcescens TA5024 obtained in Example 4
was cultured overnight on an L broth agar slant containing 100 μg/yd of kanamycin sulfate, and then
Preculture medium containing μg/- [sucrose 10%, urea 1.0
%, dipotassium phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, ferrous sulfate heptahydrate 0.001%, corn steep liquor 0.6% and calcium carbonate 1
Pour 15 d of the solution containing 15% into a 500-volume shake flask, and inoculate one platinum loop into sterilized (pH 7.0) E. Shaking culture was carried out at 30°C for 24 hours, and the preculture solution 40- obtained in this way was used as a fermentation medium [sucrose 15%, urea 1.5%,
Dipotassium phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, ferrous sulfate heptahydrate 0601%, corn steep liquor 6%, yeast extract 0.1% and calcium carbonate. 1% (pH 7,0)) was inoculated at 1.21, and incubated in a 2.41-volume jar fermenter at 30°C with 0 aeration volume.
．． Incubate for 617 minutes under agitation (750 revolutions/min), and incubate for 20 minutes.
After a period of time, L-alanine, L-cystine hydrochloride, and L-methionine are each added to a final concentration of 0.06%. 72 hours after the start of culture, d-biotin 190trt/
A culture solution containing 11 is obtained. The culture solution 101 is collected, heat sterilized, and then filtered to remove insoluble matter. Next, after adsorption on activated carbon and washing, the adsorbed material is
Elute with 0% ethanol (pH 9,0) and concentrate under reduced pressure. After the residue is dissolved in water, the pH is adjusted to 8 with aqueous ammonia, and this aqueous solution is passed through a column filled with DOWEX 1X2 (formic acid type). After washing the column with water, 0.01M
Elutes with formic acid. After collecting and concentrating the d-biotin-containing fractions, the pH was adjusted to 3.5 and left to cool overnight. The resulting crystals are collected by filtration, dried, and then recrystallized from water.
- Obtain 0.95 g of biotin.

[Brief explanation of the drawing]

Figures 1 and 2 are 98M201 plasmid D, respectively.
Restriction endonuclease cleavage maps of NA and pLGM201 plasmid DNA are shown, and FIG. 3 shows the construction of 98M201 plasmid DNA and pLGM201 plasmid DNA. happy picture number

Claims (1)

[Scope of Claims] 1. A microorganism that belongs to the genus Serratia, has resistance to substances similar to biotin structure, and has the ability to produce and accumulate d-biotin. 2, actitiazine acid or 5-(2-thienyl)-n-
The microorganism according to claim 1, which is resistant to either or both of valeric acids. 3. A microorganism in which a microorganism belonging to the genus Serratia contains a recombinant plasmid in which a deoxyribonucleic acid responsible for producing and accumulating d-biotin, which is collected from a microorganism belonging to the genus Serratia and having the ability to produce and accumulate d-biotin, is incorporated into a vector plasmid. 4. A method for producing d-biotin, which comprises culturing a microorganism belonging to the genus Serratia having the ability to produce and accumulate d-biotin in a medium, thereby producing and accumulating d-biotin in the medium, and collecting the same.