JPS6344889A - Gene to code biotin synthase and use thereof - Google Patents

Abstract

PURPOSE:To produce biotin in high efficiency, by forming DNA to code gene information participating in biosynthesis reaction of biotin from desthiobiotin derived from a bacterium belonging to the genus Bacillus, capable of producing biotin. CONSTITUTION:A recombinant vector which is integrated with NDA to code gene information participating in biosynthesis reaction of biotin from destiobiotin derived from a bacterium belonging to the genus Bacillus, capable of producing biotin is formed. A bacterium which is transformed or transduced with the recombinant is cultivated in the presence of a procursor of biotin biosynthesis in a medium containing a proper carbon source to produce biotin, which is separated and purified. A recombinant vector which is integrated with DNA in the vicinity of a promoter under the control of promoter is preferable as the recombinant vector and a bacterium belonging to the genus Escherichia coli may be cited as the bacterium for transformation.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to deoxyliponuclei III encoding genetic information involved in biotin biosynthesis reaction from desthiobiotin.
(DNA) and its use. More specifically, the present invention relates to the above DNA, a recombinant vector incorporating the DNA, a bacterium containing the recombinant vector, and a method for producing biotin using the bacterium.

(Prior Art) Biotin is a vitamin necessary for animals, plants, microorganisms, etc., and is a substance that is strongly desired to be produced industrially by applying genetic modification technology. In recent years, the genetic information involved in the biotin biosynthesis reaction has been analyzed in Escherichia coli [Journal of Bacteriology 14, 20]
65 (1967), Journal of Bacteriology-±12.830 (1972), Nature 276
．． 689 (1978), etc.], and biotin production technology using Escherichia coli is being investigated by applying genetic modification technology using the analysis results.

However, these methods cannot be used, for example, when desthiobiotin is used as a biotin precursor [Japanese Patent Application Laid-Open No. 61-149
No. 091], it is about 12μg/rrtl, and when glucose is used as a carbon source without using a precursor [JP-A-61-202686], it is about 2μg/rtft, and in both cases, biotin It has the disadvantage of low production.

On the other hand, a method of producing biotin by culturing microorganisms belonging to the genus Bacillus is also known, and some bacteria have been found that produce much more biotin than E. coli (for example, JP-A-56-160998). No. 58-
No. 60996, No. 58-152495, etc.). However, very little analysis of the genetic information involved in biotin biosynthesis in bacteria belonging to the genus Bacillus has been conducted, and therefore it has been extremely difficult to apply this information to genetic recombination technology.

(Problems to be Solved by the Invention) Accordingly, the present inventors have developed a DNA that carries the genetic information involved in biotin biosynthesis from a Bacillus bacterium using the conventional technology.
As a result of intensive studies to collect A, we found that DNA that carries the genetic information involved in the reaction to biosynthesize biotin from desthiobiotin, which is the final pathway of the hyotine biosynthesis pathway (hereinafter referred to as DNA encoding biotin synthase) It was discovered that bacteria successfully harvested and transformed with a recombinant vector incorporating the DNA have a high ability to produce biotin, leading to the completion of the present invention.

(Means for Solving the Problems) Thus, according to the present invention, as a first invention, D carrying genetic information involved in the biotin biosynthesis reaction from desthiobiotin derived from Bacillus bacteria having biotin-producing ability.
A second invention provides a recombinant vector incorporating the DNA or a promoter thereof, and a third invention provides a bacterium transformed or transduced with the recombinant vector. Furthermore, as a fourth invention, a method for producing biotin by culturing these bacteria is provided.

In the present invention, DNA encoding biotin synthase
Microorganisms used for the purpose of collecting biotin-producing bacteria belonging to the genus Bacillus, especially Bacillus sphaericus,
In other words, as long as the microorganism has an enzyme system for biotin biosynthesis, it may be a newly isolated strain from nature or an existing cultured strain. , physical treatment such as X-rays, γ-ray irradiation, or chemical treatment using drugs such as nitrosoguanidine). Furthermore, in order to increase biotin production ability, the above-mentioned bacterial strain may be made resistant to actidiazic acid (i.e., acidomycin) or 5-(2-thienyl)valeric acid, either alone or both. Specific examples of such strains include, for example, Bacillus sph.
aericus) IFO3525 and its N-
Methyl-N′-nitro N-nitrosoguanidine-treated mutants (FERM P-6422, FERM BP-
1098, FERM P-6143), etc.

DNA encoding biotin synthase from these bacterial strains
There is no need to use a particularly limited method for collecting the DNA. For example, the total DNA from the above bacterial strain is isolated and purified using a conventional method, cut with a restriction enzyme, and then the same cohesive end as the restriction enzyme is attached to the DNA. The resulting recombinant vector is used to transform (or (transduction) and select a strain endowed with biotin-producing ability from the resulting transformed strains.

Business DNA includes modified DNA that has substantially the same function as the same DNA, that is, DNA that has the same function even if a part of the base sequence has been replaced, inserted, or deleted. Of course, it is also included.

Furthermore, as the above-mentioned DNA, one containing a Shine-Dalgarno sequence, a translation start codon, and a translation stop codon is preferably used.

When constructing a recombinant vector, plasmid vectors, λ9-based phage vectors, etc. are used, but if necessary, a promoter can be simultaneously incorporated to improve the biotion production ability of transformed or transduced bacteria. Can be done.

In the present invention, any vector for constructing a recombinant vector may be used as long as it can transform (or transduce) the biotin-requiring mutant strain. For example, when E. coli is used as the biotin-requiring mutant strain,
Stringent p as an EK-based plasmid vector
scroll.

pRK353. pRK646. pRK248゜pDF4
1, Col E 1 as a relaxing type
, pVH51, pA'c105. R8F2124,
pCRl, pMB9. 'pBR313゜pBR322
, pBR324, pBR325゜1) BR327, pB
R32s, 1) KY2289゜1) KY2700.1)
KN80. DKC7゜pKs158', "pvK2o
o4. , pACYCl.

pAcycls4. puCs, 1) UO3, etc., λgt
λat, λC9λgt, λB as phage vectors
, λWES・λC1λWES・λB.

λZJv i r−λB′, λALO−λB.

λWES-T5622. λDam etc. are used. Among these vectors, plasmid vectors are particularly prized.

Furthermore, the DNA encoding biotin synthase contained in the recombinant vector has a promoter sequence that functions in the above-mentioned transformed strain. means a DNA having a promoter sequence) and must be integrated downstream thereof.

The promoter that can be used in the present invention may be either natural or synthetic, as long as the recombinant vector into which the promoter is incorporated can function in the host bacterium. Such promoters are:
j! derived from Escherichia coli! ac promoter, j! pp promoter, PL promoter, PR promoter, Trp promoter, TaC promoter, etc. when using Escherichia bacteria as a host, α-amylase promoter derived from Bacillus bacteria (e.g., Journal of Biochemistry (J, Biochem, > 9
8.

P1147, 1985: Journal of Bacteriology (J, Bacteriol,) 158. I
), 3-13 = 69.1984: G'ene" 23. p
, 267° 1983), benicillinase promoter from Bacillus licheniformis,
Trp promoter from subtilis, phage 5PO2
0.3 and 'b promoters can be used when the host is Bacillus bacteria, and the catechol-2,3-oxygenase promoter derived from Pseudomonas bacteria can be used when the host is Pseudomonas bacteria. As for natural products, those contained in the vector used as a recombinant vector may be used as is.

In addition, the biotin-requiring mutant strain may be any strain as long as the DNA carrying the genetic information of the Bacillus genus can be expressed within the bacterial body, and such mutant strains can be obtained with good reproducibility by known methods. (e.g., Journal of Bacteriol.
) 115. p662.1973: Proceedings of National Academic Science
In USA (Pro, Natl, 8 cad,
Sci, US^)69. p2219.1
972: Virology Birth 2°D
474.1970, etc.).

For example, Escherichia coli C600 [Proceedings of the Natural Academy of Sciences Vol. 1 4579 (1974)] is subjected to mutation induction treatment according to the method described in the Journal of Bacteriology 11930 (1967). A strain lacking the ability to biosynthesize desthiobiotin into biotin (hereinafter referred to as Escherichia coli 3) can be used.

The method of transformation is not limited in any way; for example, when using Escherichia coli 3 as a biotin-requiring mutant strain, it can be carried out according to the conventional method of treating the bacterial cells with calcium chloride (for example, in the journal
Of Molecular Society Biology IU, 154 (1
970)).

From among the transformants of the biotin-requiring mutant strains thus obtained, a strain newly endowed with biotin-producing ability is selected by a recombinant vector into which DNA encoding a biotin synthase derived from a Bacillus bacterium is integrated. For example, from the composition of the medium in which the biotin-requiring mutant strain of the parent strain can grow, the transformed strain is screened using a medium in which biotin has been substantially removed, and a strain that can grow in the medium is selected. Just do it.

An exemplary transformed strain thus obtained is Escherichia
The hybrid plasmid isolated from the cultured bacterial cells of Escherichia coli 3 (1) SBOl was named pSBOl. As shown in Example 1 below, this I)SBOl is a DNA fragment derived from Bacillus sphaericus and plasmid pB.
It was inserted into R322, and the restriction enzyme breakpoint map is shown in FIG. From Figure 1, the DNA that carries the genetic information involved in the biosynthesis of biotin contained in psBOl is
Starting with MboI, Hindll, Hindll
.

EcoRl, Saj! 1. HtndllI, PstI.

Approximately 8.2kb DN arranged in the order of EcoRl and BamHI
It will be understood that it is present in the A fragment (the shaded area in FIG. 1). Note that MboI in this map indicates the starting point, and does not deny the possibility that MboI exists in locations other than this location.

Of course, the recombinant vector incorporating the DNA encoding biotin synthase included in the present invention is not limited to psBOl. For example, it is possible to newly derive a plasmid incorporating the DNA from psBOI by utilizing the unique restriction enzyme cleavage site of pSBOI. [After complete degradation with C0RI, an enzymatic recombination reaction was performed, and the resulting plasmid mixture was used to transform a strain lacking the ability to biosynthesize desthiobiotin to biotin. Among the strains, a strain that has been endowed with biotin-producing ability and that has a plasmid smaller than pSBOI may be selected.

An exemplary transformed strain thus obtained is Escherichia
The hybrid plasmid isolated from the cultured strain was named psBO3. The restriction enzyme breakpoint map of DSBO3 is shown in Figure 2, and the DNA encoding biotin synthase
starts with MboI and HindT[1, Hind
l[[, EcoRI.

Approximately 4.5 kb DNA fragments arranged in the order of BamHI (second
It will be understood that this is present in the shaded area in the figure. This approximately 4.5 kb DNA fragment is the approximately 3.5 kb (F) portion of the original approximately 8.2 kb DNA fragment (
That is, the part from MbOI to EC0RI) and about 1k
Part b (i.e. EC0RI to B a rrL)
However, when 11 pieces of approximately 1 kb of DNA were separately integrated into 1) BR322 and transformed into a biotin-requiring mutant strain, it did not show biotin-producing ability. The part encoding the synthase is about -3 from MboI to EC0RI.
It was confirmed that it existed in a 5 kb fragment.

Similarly, psBO3 is added to the restriction enzyme HindI[[
A smaller plasmid was created by completely decomposing the vector with @i ndll and enzymatically recombining it with another vector cut with @i ndll, and selecting a strain endowed with biotin-producing ability from among the resulting transformed strains. It is possible to obtain strains with

An example of a transformed strain thus obtained is Escherichia coli 3 (pSBl 6). The restriction enzyme cleavage map of plasmid 1) SB16 present in this transformed strain is as shown in Figure 3, and the restriction enzyme cleavage map of the DNA fragment derived from Bacillus bacteria incorporated therein is shown in Figure 4. As shown in . From this result, the DNA encoding biotin synthase is 1ylbo:
[starts with TaqI, Rsal, Mael, MaeI
[[.

Taql, RsaI, Mbol, M, ael [[.

MboI, Hincil[, TaqI, TaQI.

It will be understood that it is present in an approximately 1.5 kb NDA fragment arranged in the following order: TaQl, Hindll.

Sequence analysis of this approximately 1.5 kb DNA fragment was performed according to the method shown in Example 3 below, and the nucleotide sequence was found to be as shown in FIG. This sequence extends from the 5'-end on the MboI side to the 3'-end on the HindllI side.
It has a large open reading frame towards the end. GTG sequence, one of the translation initiation codons of prokaryotes (Annual Review of Microbiology (Ann, Rev, formerly known as crobiol))
) ■, p 365.1981) is located at positions 282-284 from the 5' end, and is the translation stop codon TAA.
332 codons are connected to GTG from 1278th to 1280th, where . 26 upstream of this GTG
A Shein-Dalgarno sequence is present at positions 5-278.

- The amino acid sequence encoded by this DNA fragment can be determined from its base sequence as shown in Figure 5. A polypeptide having such an amino acid sequence is composed of 331 amino acids, and its molecular weight is calculated to be 36,888 daltons. In this case, methionine encoded by the transcription initiation codon GTG remains and may consist of 332 amino acids. This polypeptide functions as an enzyme that biosynthesizes biotin from desthiobiotin in bacteria.

By the way, in bacteria of the genus Bacillus, it is known that even enzymes with the same function have different amino acid sequences depending on the species (Journal of Bacteriology).
J, Bacteriol) 1ifl.

p369.1984). In these examples, some amino acids have been substituted, deleted, or inserted, and in some cases, even though the base sequence has changed, the amino acid sequence itself has not changed at all. Therefore, the base sequence of DNA encoding biotin synthase collected from species other than Bacillus sphaericus and the amino acid sequence that can be determined from that sequence may differ slightly from the base sequence and amino acid sequence shown in Figure 5. sell. Passionate, this DNA
It is also possible that the amino acid sequence or base sequence of the fragment may be artificially changed. Specifically, as shown in Example 6 below, the initiation sequence GTG is used as the initiation sequence ATG, but in any case, the polypeptide encoded in the DNA fragment is substantially the same as biotin synthase. Modifications, ie, substitutions, insertions, or deletions of amino acid sequences or base sequences, are also included in the present invention. Although the limits of modification are not necessarily uniform, it is expected that anything that hybridizes with the base sequence shown in FIG. 5 will function as a biotin synthase.

Hosts for the recombinant vectors used in the present invention include bacteria of the genus Escherichia such as Escherichia φ coli, bacteria of the genus Bacillus such as Bacillus subtilis and Bacillus sphaericus, or bacteria of the genus Pseudomonas such as Pseudomonas putida and Pseudomonas aeruginosa. can be used.

In order to transform (or transduce) these hosts and obtain transformants with excellent biothion production ability, in addition to being able to replicate in the host bacteria as described above, biotin production is required. DNA encoding genetic information involved in the synthetic reaction must be integrated under the control of a promoter that can function in the host bacterium. The promoter is incorporated upstream of the DNA, preferably within close proximity to the DNA, specifically within 300 bases, more preferably within 150 bases.

A recombinant plasmid vector containing the DNA under the control of a promoter is prepared by the following procedure. For example, pssoi, pSBO3, DSB16, I) SB10
From the plasmid containing the DNA such as 3 etc.
The region containing NA is cut with a restriction enzyme, for example, Tr.
When using the p promoter, pDR720 (Ge
This cut DNA may be inserted downstream of the promoter sequence of ne20°231 (1982)). For the host Escherichia bacteria, Escherichia coli C6
00, same JM103. Same MC169, CAGl 138
．． JE8476, GC4670゜AB1899, JE8
Not only bacteria frequently used in the field of genetic engineering such as GW5229 and GW5229, but also strains newly isolated from nature or existing cultured strains may be used, and these strains can be treated with ordinary microbial mutagenesis methods. E. coli strains with weak intracellular protease activity, such as MCl69, are particularly preferred. As an example of the recombinant vector thus obtained, psB3 is shown in FIG.
01, and it can be seen that the DNA encoding biotin synthase is integrated under the control of the Trp promoter and adjacent thereto.

In addition, when the host is a bacterium of the genus Bacillus, the promoter may be, for example, α-amylase of Bacillus licheniformis or Bacillus stearothermophilus, which is thought to generally function in bacteria of the genus Bacillus.
You can use promoters, etc. In addition, when connecting the above-mentioned DNA to these promoters, the DNA fragment containing the translation initiation codon GTG sequence shown in FIG.
In order to make the 3' sequence identical to the 3' sequence of the promoter, a synthetic linker or the like may be used as shown in Example 6. Of course, when using a Bacillus bacterium as a host, the completed recombinant vector must be able to replicate in the host, and for this, a part of the vector must contain a Bacillus bacterium such as pLJBl 10. It must contain the replication initiation region of a plasmid that is generally considered to be capable of replication.

As for the host bacteria of the genus Bacillus, of course bacteria classified as Bacillus subtilis, which are often used in the field of genetic engineering, are collected, as well as genetic information involved in the biotin biosynthesis reaction, such as DNA encoding the same substance synthase. All biotin-producing bacteria belonging to Bacillus sphaericus that can be used for the purpose of the present invention can be used for the purpose of the present invention. An example of the recombinant vector thus obtained is pBHB13 shown in FIG.

This vector has a DNA encoding pyotin synthase integrated under the control of the α-amylase promoter derived from Bacillus licheniformis, and pUBl
It can be understood that this has a lo replication initiation region.

Similarly, when the host is a Pseudomonas bacterium,
A catechol-2゜3-oxygenase promoter or the like is used as the promoter, and plasmid R8F1010 and its derivative [in.
Blasmes of Medical, Environmental and Commercial Invoices (In Pl.
asmids of Medical, Environ
mental and CommercialInpo
rtance), p411.1979].

In the present invention, the recombinant vectors obtained by the method described above are used to transform the corresponding host bacteria to obtain transformants. For example, when transforming Bacillus sphaericus, the method described in Journal of General Microbiology 130.203 (1984
Transformation may be performed according to the method described in ).

The present invention also provides a method for producing biotin, which involves culturing the transformant thus obtained to produce biotin, and separating and purifying the produced biotin. When producing biotin, the biotin precursor to be added to the medium used during culture differs depending on whether the host cell to be transformed using the recombinant vector has the ability to produce desthiobiotin. For example, if a bacterium of the genus Escherichia that does not substantially have the ability to produce desthiobiotin is
When producing biotin by culturing a transformant transformed with sB301, it is necessary to add desthiobiotin to the medium as shown in Example 7. on the other hand,
When using as a host bacteria such as Bacillus sphaericus 1FO3525 and Bacillus sphaericus NCl89370, which can assimilate desthiobiotin precursors such as bimelic acid and azelaic acid to produce desthiobiotin, There is no need to add desthiobiotin, and it is sufficient to simply add vimelic acid or azelaic acid as a desthiobiotin precursor to the medium.

The amount of desthiobiotin or its precursor to be added is not particularly limited, and may be appropriately selected depending on the biotin-producing ability of the host cell.

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, mandose, mannose, starch, starch hydrolyzate, and molasses can be used. Nitrogen sources include ammonia, various inorganic and organic ammonium salts such as ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium carbonate, and ammonium acetate, meat extract, yeast extract, corn staple liquor, casein hydrolyzate, and defatted soybean meal. Natural organic nitrogen sources such as carbon dioxide and its digested products can be used. Many natural organic nitrogen sources can be both nitrogen and carbon sources. Furthermore, inorganic substances used include potassium hydrogen phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, calcium chloride, zinc chloride, copper sulfate, manganese chloride, cobalt chloride, ammonium molybdate, and sulfuric acid. can.

In addition, if the created microorganism is endowed with antibacterial drug resistance, by adding the corresponding antibacterial agent to the culture medium, it is possible to prevent the plasmid DNA that has the antibacterial drug resistance possessed by the microorganism from being shed as a marker. Contamination with contaminating bacteria can be prevented.

Furthermore, if part or all of the reaction in the process of forming biotin from the desthiobiotin precursor is inhibited by biotin in the medium, it is preferable to use a medium from which biotin has been removed. Furthermore, in order to avoid the feedback repression effect caused by biotin, it is preferable to add the desthiobiotin precursor at the end of the logarithmic growth phase of the bacterial cells (see Japanese Patent Publication No. 61-3295'9).

Cultivation is preferably carried out under aerobic conditions such as shaking culture or aerated agitation culture, usually at 20 to 45°C for 1 to 5 days.
It will be held for about a day.・In this case, conditions are required for the promoter that transcribes the biotin synthase gene to function appropriately. For example, when using the Trp promoter in Escherichia bacteria, ・By adding indole acrylic acid, the transcriptional activity of the promoter can be increased. It is possible to induce

(Effects of the Invention) Thus, according to the present invention, a DN encoding a biotin synthase derived from a Bacillus bacterium having biotin-producing ability.
By providing the A fragment and transforming (or transducing) bacteria with a recombinant vector incorporating the DNA fragment, bacteria with excellent biotin-producing ability can be obtained, and these bacteria can be further cultured. A method for efficiently producing biotin will be provided.

(Example) The present invention will be described in more detail with reference to Examples below.

Example 1.9 (1) Preparation of total DNA fragments Bacillus sphaericus IFO3525 resistant strain in combination with aquithiazine acid and 5-(2-thienyl)-valeric acid (F
ERM BP-1098) and 11 LB medium (Bactodryptone 1%, Yeast Extract 0.5%, N
aCJ! 1% glucose, 0.1% glucose (adjusted to pH 7.5), shaking culture was carried out at 28° C. for about 5 hours, and the bacterial cells were collected by centrifugation in the logarithmic growth phase. From this bacterial cell, the phenol method [Biochem Biophys Acta (Bioche
m.

B111) he's, Acta) L2.619~.
Total DNA was extracted by 1 in 1973, I! Finally, total DNA3. OIRg was obtained.

500 μq of the obtained total DNA was taken, restriction endonuclease MboI was added, and the reaction mixture was allowed to react at 37°C for 30 minutes to partially cleave it.Then, the reaction product was subjected to agarose gel electrophoresis, and DNA fragments ranging from about 4 kb to about 10 kb were recovered. .

(2) Cleavage of vector pBR322 pBR with ampicillin resistance and tetracycline resistance as markers
322 (commercial product, Pharmacia Ia) was completely degraded with restriction endonuclease 3 μm HI, and then treated with alkaline phosphatase.

(3) Recombination reaction (1) The prepared DNA fragment (0.1 μg) and the cleaved pBR322 (0.1 μg) prepared in (2)
) with 10 m HMO (, J!, T4 DNA ligase 0.0.
A hybrid plasmid in which the DNA fragment was integrated into I) BR322 was obtained by mixing with 3 units and reacting overnight at 4°C.

(4) Introduction of hybrid plasmid into Escherichia coli [)Hl [Molecular Cloning A Laboratory Manual (Nolecul)]
ar Cloning A, Laboratory
Manual) p.

505.1982] in 100 ae of φ medium (yeast extract 0.5%, Bactodrypton 2%, MG
SO40.5%: adjusted to EIH7.6) at 37℃ about 5
Perform a shaking culture for a period of time, centrifuge the bacterial cells during the logarithmic growth phase, and
Collected by t. This bacterial cell was treated with TFB I solution (30111
8 Potassium acetate, 100mMRbCj! , 10 mHCa
CJ! 2.50mMnCJ, 15% glycerin:
Adjust the pH to 5.8) and wash with 401d, and wash the resulting bacterial cells with trb m solution (10mHMOP8.75mHCaCj!).
2.10mMRbCO, 15% glycerin; pH 6.5
(adjusted to) 10 m and left at 0° C. for 90 minutes to obtain competent cells.

0.1 μj of the hybrid plasmid obtained in (3) was added to 0.2 m of this permissive cell suspension and allowed to stand at 0° C. for 30 minutes. After keeping at 42°C for 90 seconds, φ medium 0.8#d! was added and cultured at 37°C for 1 hour, and 0.1
- was spread on a 1.5% agar medium of LB medium containing 100 μSF/me ampicillin, and cultured overnight at 37°C.

Next, about 2.50011 colonies that appeared were picked with a toothpick, inoculated onto a 1.5% agar medium of LB medium containing 15 μg/Idl tetracycline, and cultured at 37° C. overnight. From this, it can be grown on a medium containing ampicillin.
Approximately 1,000 ampicillin-resistant and tetracycline-sensitive strains, which were able to grow on a medium containing tetracycline-33-, were obtained.

(5) Isolation of hybrid plasmids 1 The thus obtained ampicillin-resistant, tetracycline-sensitive strains were divided into 5 groups (each group containing about 200 different hybrid plasmids) and incubated in 50 d of LB medium containing 50 μ9 of ampicillin. The cells were incubated with shaking at 37° C. overnight, and the bacterial cells were collected by centrifugation. From this bacterial body, Birnboim and Dory
ly) method [Nucreitsk Acid Research (Nu
cleic Ac1d Res, > 7.1513~
．． (1979)] to extract plasmid DNA. The plasmid DNA thus obtained was added to the vector pBR322 with Bacillus sphaericus IF.
Various NA fragments obtained from Q3525 were inserted, and the average length of the inserted DNA fragments in this hybrid plasmid DNA group was about 6 kb.

(6) Introduction of hybrid plasmid into biotin-auxotrophic E. coli Escherichia coli C600 to Pai and Lichstein's method [Journal of Bacteriology (J, Bacteri.
ol) 94, p1930.1967] N-
A mutant strain of Escherichia coli lacking the ability to biosynthesize desthiobiotin (DTB) to biotin was obtained by mutation induction treatment with methyl-N'-nitro-N-nitrosoguanidine. This is 1001111! in LB medium at 37°C
The cells were cultured with open shaking for about 2 hours, and the cells were collected by centrifugation at the logarithmic growth phase. This bacterial body is called Kushner.
r) method [Genetics Engineering (a
enetic Enoineerina) p.

17.1978] and were considered permissive cells.

Next, the hybrid plasmid DNA group obtained in (5) was added to each group by 0.1μ9 of the above permissive cell suspension.
In addition to 2 d, the tube was allowed to stand at 0°C for 30 minutes.

After keeping at 43.5°C for 30 seconds, LB medium 1.8- was added and cultured at 37°C for 1 hour, followed by centrifugation to collect bacteria. After washing the bacterial cells twice with 85% NaCf, they were suspended in 85% NaCf111d. Next, 0.1 d of the bacterial cell suspension was added to O containing 50 μg/Id ampicillin.
A medium [glycerin 2%, Casamino II (vitamin free) 1%, K2 HP'040.2%, KH2PO4
0.1%, MGSO4.7H200,005%, FeSO4.7H200,001%, MnSO4-4-6820 0.001%, thiamine hydrochloride 0.00001%] on a 1.5% agar medium,
The cells were cultured at 37°C for two nights. The two colonies that emerged were ampicillin resistant and non-biotin auxotrophic, thus containing DNA carrying genetic information involved in the biosynthesis of biotin.
Two transformed strains using the hybrid plasmid incorporating A were obtained.

(7) Hybrid plasmid analysis The two transformants obtained in (6) were placed in CA medium 5#Il! containing 50μ9/rd ampicillin! After culturing with shaking overnight in a microcentrifuge and collecting bacteria by centrifugation, hybrid plasmid DNA was extracted by the method of Birnboim and Doll, described above.

Hybrid plasmid DNA obtained from each strain
A has a total chain length of 12.5 kb, approximately 8.2 kb
A DNA fragment was inserted. For these two hybrid plasmid DNAs, various restriction endonucleases (BamHI, )1indl[[, PStI, E
coRI, 3aj! When cleavage patterns according to I) were prepared and compared, it was found that the two hybrid plasmid DNAs were identical because they had exactly the same patterns. Furthermore, this hybrid plasmid is BQJ! It was also confirmed that there were no cut points caused by II, xhO, or KDnI. This hybrid plasmid DNA was named I) SBO1, and its restriction endonuclease cleavage map is shown in FIG. In addition, the first
The numbers in the figure indicate approximate values of bp.

Next, the DNA inserted into psBO1 plasmid DNA
Fragment A is the DN of Bacillus sphaericus IFO3525
Southern hybridization was performed to prove that it was an A fragment. First, the total DNA extracted and purified in (1) was cleaved with restriction endonuclease EcoRI, and the product was subjected to agarose gel electrophoresis using the Southern method [Journal of Molecular Biology (J. Ho1. Rio
t,) 98, 1) 503.1975], it was transferred to Nido 0 Cell O-Sfilter. Apart from this p
One micrometer of $801 plasmid DNA was labeled with P and used as probe DNA. Preparation is by Riaby
) et al. [Journal of Molecular Biology (J, Ho1. Biol) 113. p2
114X10 CDm/μ9 DNA by nick translation according to [37.1977]
A probe DNA having a specific activity of . Using 0.1 μg of this probe DNA, hybridization was performed on the previously prepared nitrocellulose filter according to the following procedure. First, filter and SSC (0
, 15MNaCJ! .

0.015M sodium citrate) 6x concentrate and oe
nhardt solution (0.02% bovine serum albumin, 0.02% bovine serum albumin,
Preliminary hybridization buffer 2 consisting of 0.02% polyvinyl pyrrolidone, 0.02% Ficoll)
20μg/lli of salmon sperm DNA heat-treated to 0d!
Preliminary hybridization was carried out at 65° C. for 4 hours using the resulting mixture. after that,
6x concentrated solution of SSC and Denhardt solution, 0.5% S
20m hybridization buffer consisting of DS
Heat-treated salmon sperm DNA was added to the solution at a concentration of 50 μ9/rd, 0.1 μ9 of heat-treated probe DNA was further added, and hybridization was carried out at 67° C. overnight. The filter was then thoroughly washed with hybridization buffer, further washed in a 6x concentrated solution of SSC at 65°C for 30 minutes, and washed in a 2x concentrated solution of SSC at 65°C for 30 minutes, repeated twice, air-dried, and autoradiated. I graphed it. As a result, it was confirmed that there was a fragment that reacted with this probe DNA in all the DNA, and the approximately 8.2 kb inserted DNA fragment in the pSBO1 plasmid DNA was proved to be a DNA fragment of Bacillus sphaericus IFO3525. Ta.

On the other hand, using the Escherichia coli biotin operon as a probe, the method of Gibbs et al. [Proceedings of National Academy of Sciences in USA (Proc, Natl.

Acad, Sci, USA), p3365.1
984], Bacillus sphaericus I FO
For 3525 full [)NA EcoR cutting 5Ili piece,
Southern hybridization was performed, but the DNA
They did not hybridize even under the conditions under which hybridization would occur if there was complementarity between them. Therefore, the complementarity between the biotin operon of Escherichia coli and the DNA of Bacillus sphaericus is extremely low. Therefore, 1) insertion of SBO1. The DNA fragment turned out to be very different from that of Escherichia coli.

(8) Confirmation of reproducibility of transformation 1) Re-transform 3 into Escherichia coli by the method described in (6) using SBO1 plasmid DNA = 40
- When transformed, all three were ampicillin resistant to Escherichia coli carrying psBOl as a plasmid.
Biotin is not required. This indicates that psBO1 plasmid DNA possesses at least a DNA fragment of Bacillus sphaericus I'FO3525, which has genetic information that can complement Escherichia coli's lack of ability to biosynthesize desthiobiotin to biotin in 3. There was found.

This pSBO1SBO1 plasmid DNA was named Escherichia coli 3 (psBOl). In addition, this bacterium is FE
It has been deposited with the Institute of Fine Technology as RM BP-1099.

Example 2゜(1) Reconstruction of hybrid plasmid The psBO1 plasmid DNA obtained in Example 1 was completely digested with the restriction endonuclease EC0RI, and the three types of D
1 μ9 of the mixture of NA fragments to 10 mHMQCj! 2.1+
T4 in 50 mH t-Lis-HCl of Elf (7,4) containing 118 ATP and 10 mH dethiothreitol.
Religion was performed by mixing with 3 units of DNA ligase and reacting overnight at 4°C.

(2) Transformation of biotin-auxotrophic E. coli T4 described above
A transformed strain using the hybrid plasmid was obtained in the same manner as in Example 1 (6) except that 0.1 μ9 of the DNA ligase reaction mixture was used.

(3) Hybrid plasmid analysis The transformant obtained in (2) was added to 5 #ll of CA medium containing 50 μg/rd ampicillin! After culturing with shaking overnight in a microcentrifuge and collecting bacteria by centrifugation, hybrid plasmid DNA was extracted by the method of Birnboim and Doorley described above. The smallest of the extracted plasmid DNAs has a total chain length of 8
．． 8Stb, pSBO1SBO1 plasmid DNA
A, the 7 kb EC0R1 cleavage fragment was missing. This hybrid plasmid was named pSBO3, and its restriction endonuclease cleavage map is shown in FIG.

3 to Escherichia coli that owns this pSBO3 plasmid DNA (psBO3)
It was named. (FERM P-8369).

Example 3 (1) Reconstruction of hybrid plasmid 3 μg of psBO3 plasmid DNA obtained in Example 2 was completely digested with restriction endonuclease Hindll. Separately, I) BR3221μ9 was added to restriction endonuclease H
Completely decomposed with indl[[. Next, 1) A mixture of three types of DNA fragments obtained by cutting SBO3 (1 μg)
pBR322<1u9) cleaved with 10 mHMaC
j! A reaction mixture was prepared by mixing with 3 units of T4 DNA ligase in 50IIIM Tris-HCl, pH 7.4, containing 2.1 mHATP and 1Qn+H dethiothreitol. A hybrid plasmid was obtained by reacting each of these overnight at 4°C.

(2) Transformation of biotin-requiring Escherichia coli A transformed strain using the hybrid plasmid was obtained in the same manner as in Example 1 (6) except that 1 μ9 of the above reaction mixture was used.

(3) Hybrid plasmid analysis The transformant obtained in (2) was added at 50μg/1lEi! After overnight shaking culture in CA medium 5d containing ampicillin and harvesting by centrifugation, hybrid plasmid DNA was extracted by the method of Birnboim and Dory described above. The smallest of the extracted plasmid DNAs had a total chain length of 6.2 kb. This plasmid contains approximately 2.0k of I) SBO3 DNA.
bh+ndm cleavage fragment was inserted, and psB
It was named 16. The restriction endonuclease cleavage map is shown in FIG.

Bacillus sphaericus IF contained in this inserted fragment
The DNA fragment derived from 03525 is MboI to H+ nd
The detailed restriction endonuclease cleavage map is shown in FIG. 4.

To further investigate this approximately 1.5 kb DNA fragment, M.
Metzing et al.'s method using phage 13 [Gene (G
ene)19. I) 269.1982.

Science 214. l)12
Sequence analysis was performed according to 05.1981. Its base sequence is as shown in Figure 5, and it has one large open reading frame from the 5' end on the MboI side to the 3' end on the HindllI side, specifically from the 5' end to the 282nd 332 codons are connected following the translation initiation codon GTG at position 284. Furthermore, by comparing the nucleotide sequence upstream of the translation initiation codon with the known sequence of 168 ribosomal RNA for Bacillus subtilis, we were able to confirm that the sequence from positions 265 to 278 corresponds to the Sheain-Dalgarno sequence. .

In addition, 3 for Escherichia coli that has pSB16 plasmid DNA and 3 for Escherichia coli (psB16)
It was named.

Example 4 Escherichia coli C600 (pB
R322) was treated with G containing 50 μg/d ampicillin and 10 μSF/d DL-desthiobiotin.
P medium [glycerin 2%, proteose peptone 3%
, Casamino Acid (Vitamin Free) 0.5%, K HP
O40.1%, KCjt 0.05%, MnSO4・
, 7) 10 0.05%, MnSO4・4~6H
O0,001%, Fe50 ・7 days 200.001%,
Thiamine hydrochloride 0.000002%, pH 7.0]3-
The cells were each inoculated and cultured with shaking at 37°C for 4 days.

After culturing, the culture solution 1d was centrifuged to precipitate the bacterial cells, and the supernatant was transferred to a test tube. Next, this supernatant was heated to 120°C.
After treatment for 30 minutes with Lactobacillus blankrum (L
actobacillus plantarumATC
Biotin accumulated in the supernatant was quantified using a microbial assay method using C8014). Table 1 shows the amount of biotin accumulated by each strain.

From the results shown in Table 1, it is clear that the microorganism of the present invention accumulates a large amount of hyotine in the medium compared to Escherichia coli C600 (DBR322).

47 Example 5゜(1) Construction of hybrid plasmid osB103 The same procedure as Example 3 was performed except that 1) UO3 (commercial product, manufactured by Pharmacia) was used as a marker for ampicillin resistance in place of pBR322 used in Example 3. An experiment was conducted in the same manner, and approximately 2. Okb's DNA
A hybrid plasmid (psB103) with a total chain length of 4.6 kb into which the fragment had been inserted was obtained (see Figure 6). This p
Escherichia coli 3 transformed by SB103 was named E. coli 3 (pSB103).

(2) Construction of hybrid plasmid psB301 (see Figure 6)
The following operation was performed to directly connect the o3 gene. ps
After completely digesting B103 with restriction endonuclease MaeI, the digested DNA fragment (2,0/lzg) was dissolved in 30mM sodium acetate (pH 4,5) and 250mHN.
aCj! , 8 in 1mM ZnS0 4.5% glycerol
Both ends of the DNA fragment were made blunt by mixing with 1 unit of nuclease and reacting at 37°C for 30 minutes.

The reaction was then subjected to agarose gel electrophoresis for approximately 2,
A 1 kb DNA fragment was recovered.

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

Approximately 2.1 kb DNA fragment (0.1 μg) recovered from agarose gel and cleaved pDR720 (0.1 μg)
) to 10mHMQCj! , 1mNATP and 10
501 of DH7,4 containing IIIM dethiothreitol
A reaction mixture containing the hybrid plasmid was obtained by mixing with 2 units of T4 DNA ligase in 118 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 Part 1 (6) except that 0.2 μg of this reaction mixture was used.

The obtained transformed strain was cultured with shaking overnight in LB medium 3d containing 50 μg/d ampicillin, and the cells were collected by centrifugation.
Hybrid plasmid DNA was extracted by the method of Birnboim and Doorley.

The smallest of the extracted plasmid DNAs had a total chain length of 6.1 kb. In this plasmid, the inter-translation start codon GTG of the biotin synthase gene derived from Bacillus sphaericus began approximately 70 bases downstream of the transcription start site of the Trp promoter. This is psB301
It was named.

Handel and SEGA (formerly AA) method and Journal of Molecular Biology (
J, Ho1. Biol, ) 53. p154.1970
According to Escherichia Coli MC169 [Journal of Bacteriology, 143. p852.1
980] with pSB301, and the resulting transformant was transformed into Escherichia coli MC169 (psB301
), Example 6 The following operation was performed to directly link the biotin synthase gene to the α-amylase gene promoter region of Bacillus licheniformis.

(1) Construction of pBHB10 psB103 was purified using restriction endonuclease Hindl [
After complete digestion, the reaction product was subjected to agarose gel electrophoresis, and a b(7) DNA fragment of approximately 2.0 was recovered. Restriction endonuclease Mael was added to 2.0 μg of this DNA fragment, and the reaction was carried out at 45° C. for 10 minutes to partially cleave it.The reaction product was then subjected to agarose gel electrophoresis, and a DNA fragment of approximately 1.2 kb was recovered.

On the other hand, the oligonucleotide fragment BbOj! as shown in FIG. -1 and BbQj! -2 was obtained. Among these, Bboj! -1 fragment 1μ3 to 5
0mHt-Lith hydrochloric acid (pH 7,6), 10m) 4Ma
Cj! The 5'
- After phosphorylating the terminal hydroxyl group, BbQJ! 1 μq of -2 fragment was added and annealed.

Next, I) U with ampicillin resistance as a marker
Cl3 (commercial product, manufactured by Pharmacia) was completely degraded with restriction endonucleases BamHI and Hindl, and then treated with alkaline phosphatase.

1μ of the cleaved approximately 2.7kb pUcl8 DNA fragment
Synthetic fragment e10mH previously annealed with 9
MOCj! , 1 mHATP# was mixed with 2 units of T4 DNA ligase in 50 mM Tris-HCl, pH 7.4 containing 10 mH dethiothreitol, and incubated at 15°C for 2 hours.
After reacting for an hour, the reaction product was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 2.7 kb was recovered. Next, the recovered DNA fragment (0.4 μg) and the previously prepared approximately 1.2
kb DNA fragment (0,015 μg) was mixed with 2 units of T4 DNA ligase in a reaction solution with the same composition as above,
The reaction was carried out at 15°C for 3 hours.

A transformed strain using the hybrid plasmid was obtained in the same manner as in Example 1 (6) except that this reaction mixture was used.

The obtained transformant was cultured with shaking overnight in 3 d of LB medium containing 50 μg/Id ampicillin, and after harvesting by centrifugation, hybrid plasmid DNA was extracted by the method of Birnboim and Dory.

Each extracted plasmid DNA was completely digested with restriction endonuclease BamHI and )lindl[[, and then subjected to agarose gel electrophoresis.
Approximately 1.3 kb containing a DNA fragment of approximately 1.2 kb prepared from
Approximately 4.kb DNA fragment and two 2.7kb fragments derived from pLICl 8. Plasmid DNA of Okb was obtained. This was named pBH810.

(Construction of 21D LJ C18-AP After completely digesting pUc18 with restriction endonuclease NdeI,
The cleaved DNA fragment (2.0 μ9) was dissolved in 3Q mH sodium acetate (pH 4,5), 250 mH NaC, 1
Both ends of the DNA fragment were made blunt by mixing with 1 unit of 81 nuclease in mM ZnSO4, 5% glycerol and reacting at 37°C for 30 minutes.

Then, the reactant (1,0 μg) was added to 10 mNMgCJ!
The mixture was mixed with 5 units of T4 DNA ligase in 50 mH Tris-HCl at DH 7.4 containing 1+HATP and 10 mM dethios L/itol, and cyclized again by reacting overnight at 4°C.

This reaction mixture was used to transform Escherichia coli C600 according to the method of Mandel and Higa. Plasmid DNA extracted from a transformant grown on an LB agar medium containing 50 μg/d of ampicillin was DLJCI
It is a plasmid of approximately 2.7 kb in which the NdeI cleavage site was removed from pUcl BN, and was named pUcl BN.

This pLJCl 8-N was subjected to restriction endonuclease EC.
The fragment obtained by complete digestion with 0RI and PStI and the approximately 1.1 kb DNA fragment having the EcoRI-PstI cleavage site of Bacillus licheniformis and containing the α-amylase gene promoter region were combined with 10 mM MOCJ.
! , 1mM ATP and 10mM buthiothreitol pH 7.4 mixed with 1 unit of T4 DNA ligase in 5011IM Tris-HCl buffer, pH 7.4,
The reaction was carried out at ℃ for 2 hours.

This reaction mixture was used to transform Escherichia coli C600 according to the method of Mandel and Higa. The transformed strain grown on LB agar medium containing 50 μg/Id of ampicillin was cultured with shaking overnight in LB medium 3d containing 50 μg/Id of ampicillin, and after harvesting by centrifugation, the method of Birnboim and Dory was used. Plasmid DNA was extracted. Each extracted plasmid DNA was completely digested with restriction endonucleases EC0RI and PstI, and then subjected to agarose gel electrophoresis.
Approximately 1.1 kb DNA fragment derived from Licheniformis and D
Approximately 3.8 kb plasmid DNA consisting of an approximately 2.7 kb DNA fragment derived from tJCl 8-N was obtained. This is p
It was named Uc18-AP.

+3) Construction of DB HB 11 1) After completely degrading BH810 with restriction endonuclease Hindl, restriction endonuclease NdeI was added to the cut DNA fragment 6.0 μ9, and the reaction was carried out at 37°C for 5 minutes to partially cut it. Then, the reaction mixture was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 1.3 kb was recovered.

This DNA fragment (0.1 μg) and puc18-AP
using the restriction endonucleases NdeI and HindI [[
The DNA fragments obtained by complete digestion with 10mHMg
Cj! , l + HATP and 10 mH dethiothreitol), pH 7, 4 (7), mixed with 1 unit of T4 DNA ligase in 50 mH Tris-HCl and incubated at 15°C.
, and reacted for 2 hours.

This reaction mixture was used to transform Escherichia coli C600 according to the method of Mandel and Higa. Ampicillin 50 μg/d [LB medium 3III containing 50 μg/Id ampicillin for transformed strains grown on B agar medium! ! After culturing with shaking overnight in
A was extracted. After completely digesting each extracted plasmid DNA with restriction endonucleases EC0RI and Hindll, it was subjected to agarose gel electrophoresis, and an approximately 2.4 kb DNA fragment containing the promoter region of the α-amylase gene and the biotin synthase gene was found. pL
Approx. Plasmid DNA of Okb was obtained.

This was named pB)IBII.

The DNA sequence was analyzed by the method of Metssing et al. using M13 phage, focusing on the junction between the α-amylase gene promoter region and the biotin synthase encoding region on pBHBl 1. The base sequence is number 9
As shown in the figure, α of Bacillus licheniformis
- An open leap ink frame (0,R) encoding Bacillus sphaericus biotin synthase in the amylase gene promoter sequence, the Shein-Dalgarno (SD) sequence, and the translation initiation codon ATG.

It will be understood that the sequence from F) except for the translation initiation codon GTG is linked.

+4) Construction of pB HB 13 1) Plasmid ptJB1 that is replicable in BH811 and Bacillus and has kanamycin resistance as a marker
10 (Journal of Bacteriology (J, B
acteriol, > 134, P318.1978
] was completely digested with restriction endonuclease EC0RI, and then the cleaved pBH810' (0.2 μg) and the cleaved pUBl 10 (0.2 μg) were added to 10 mHMQ.
C,e, 1aHATP and 10mM buthiothreitol 1) 87.4T in 50mH Tris-HCl
The mixture was mixed with 2 units of 4 DNA ligase and reacted at 15°C for 2 hours.

This reaction mixture was used to transform Escherichia coli C600 according to the method of Mandel and Higa. Ambishiri, N50μ9/atl! Approximately 400 colonies grown on LB agar medium containing kanamycin 10μ9/d
! When replicated on an LB agar medium containing 52 strains, 52 transformants showing ampicillin resistance and kanamycin resistance (Km) were obtained. LB medium containing 10μ3/me of kanamycin each! After culturing with shaking overnight in a microcentrifuge and collecting the bacteria by centrifugation, plasmid 11) NA was extracted by the method of Birnboim and Dory. The smallest of the extracted plasmid DNAs has a total length of approximately 9.5k.
It was b. This plasmid consists of pB, HB11 and pU.
This is a hybrid plasmid consisting of Blo, a shuttle vector that can be replicated in both Escherichia and Bacillus, and was named pBH813 (see Figure 8).

(5) Transformation of Bacillus sphaericus Bacillus sphaericus NGIB9370 was added to 50Id of PB medium (Daf)/antibiotic medium No. 3
1. , 75%) at 37° C. until the mid-logarithmic growth phase, and the bacterial cells were collected by centrifugation. This bacterial body is S
MMP solution [2XSMM (IM sucrose, 4QmH sodium malate, 40mHMQCj! ・6H20, pH6
, 5) mixed with equal amounts of 4XPB (Difco Antibiotic Medilum No., 37%)]
was suspended in 5 m of a solution containing 1% BSA, and acetyl muramizois was added at a rate of 0.3 towers/ae.
The reaction was carried out at 37°C for 45 minutes. After collecting the bacterial cells by centrifugation, they were washed twice with SMMP, and 1afSMM
The cells were suspended in P to prepare a protoplast solution.

1 μg of DBH813 prepared from Escherichia coli was added to 0.51 d of this protoplast solution, and 1.5-4
0% PEG6000 (polyethylene glycol 600
0) Add 2XSMM50d to 40g, dilute to 100ae with distilled water), suspend gently, and leave at room temperature for 2 minutes. After diluting by adding SMVP, the bacteria were collected by centrifugation. BSA is 1
After suspending in 1 mg of SMV P containing 30%
After statically culturing at ℃ for 1.5 hours, this culture solution was 0.1#! l! DM4 medium containing 150 μ9/d kanamycin [agar 0
．． 8%, Casamino No. 5%, East Extract 0.5%, 0.33M Kohachi! (DH7,3), 2HPO40.35%, KH2PO40.15%, glycerin 0.5%, 0.02M Moc1.0.01%B was smeared on SAI, and cultured at 37°C for 3 days to 1 week. .

Next, the colonies that appeared were picked out using toothpicks, and 10g
The cells were inoculated into GP medium containing /d kanamycin, cultured with shaking overnight at 37°C, and the bacterial cells were collected by centrifugation.

Plasmid DNA was extracted from this bacterial cell by the method of Birnboim and Doorley. When the restriction endonuclease cleavage pattern of the plasmid [)NA thus obtained was examined, it was found that pB
It showed the same pattern as HB13, and it was confirmed that I) BHB13 was introduced into Bacillus sphaericus NCIB9370. However, the restriction endonuclease 13 amHI cleavage site present on pBHB13 is modified by methylase within Bacillus sphaericus NCIB9370 cells and is therefore not cleaved by restriction endonuclease Bam) II. Bacillus sphaericus NCIB9 transformed with pBH813
370 to Bacillus sphaericus NCIB9370 (
pBH813).

In addition, Bacillus sphaericus IFO3525 was used instead of Bacillus sphaericus NCIB9370, and Bacillus sphaericus NCIB9370 (pBHB13
The transformed strain obtained by the same method as above except that it was transformed with pBHB13 extracted from B.

Bacillus sphaericus N0IB9370 and Bacillus sphaericus IFO3525, which were similarly transformed with pUBI 10, were named Bacillus sphaericus NCIB9370 (pLJBllo) and Bacillus sphaericus IFO3525 (pLJBllo), respectively.

Example 7 Two strains of Escherichia coli MC169 (pSB301) and Escherichia coli M0169 (pDR720) having pDR720 as plasmids were treated with 50 μg/1 tdl of ampicillin and 100 μ9/1
d PC medium containing DL-desthiobiotin (GTB) (2% glycerin, 5% proteose peptone, 2% casamino acids, 0.1% K2HPO4, KCj!0.05
%, Mo2O3・7HOO,05%, MnSO4・4~6HOO,001%, FeSO4・7H200,001
%, pH 7.0 > 1.2 times concentration medium 3d, and after culturing with shaking at 37°C for 1 hour, 16.7μ of indole acrylic acid was added to induce the Trp promoter.
A total of 97 udl was added, and the culture was further incubated overnight at 37°C with shaking.

After culturing, 1 m of the culture solution was centrifuged to precipitate the bacterial cells, and the supernatant was transferred to a test tube. Next, this supernatant was heated to 120°C.
After treatment for 30 minutes, biotin accumulated in the supernatant was quantified by microbial immobilization using Lactobacillus plantarum. Table 2 shows the amount of biotin accumulated by each strain.

From the results shown in Table 2, it is clear that the microorganism of the present invention accumulates a large amount of biotin in the medium compared to Escherichia coli MC169 (DDR720).

Note that the biotin accumulation amount of the microorganism according to the present invention is based on various biotin-producing mutant strains of Escherichia coli (for example, JP-A-61-149091, JP-A-61-20268).
This value is high even when compared to No. 6), which indicates the usefulness of the gene involved in the production of phlegm, which is derived from Bacillus genus Bacteria and is related to sleep.

Example 8 Bacillus sphaericus NCIB9370 (pBHB1
3), Bacillus sphaericus NCIB9370 (p
LJBl 10), Bacillus sphaericus IFO35
25 (pBH813) and Bacillus sphaericus IF
Four types of strains of O3525 (pUBl 10) were inoculated into GP medium 3d, and after culturing with shaking at 28°C overnight, DL-DTB was added at a concentration of 25 μg/- at the end of the logarithmic growth phase.
or vimelic acid was added at a concentration of 500 μg/d, and cultured with shaking at 37° C. for 2 days.

After culturing, the culture supernatant was assayed for biotin using Lactobacillus blankrum using a microbial emplacement method. Table 3 shows the amount of biotin accumulated by each strain and precursor.

- 66 ~ From the results in Table 3, it will be understood that the microorganism of the present invention produces biotin using vimelic acid as a precursor in the same way as when DL-TB is used as a precursor.

[Brief explanation of drawings]

Figures 1 to 3 are pSBO1 plasmid DNA.
, shows the restriction endonuclease cleavage maps of psBO3 plasmid DNA and DSB16 plasmid DNA, and FIGS. 4 to 5 show the restriction endonuclease cleavage maps, nucleotide sequence, and amino acid sequence of the DNA fragment derived from Bacillus bacteria inserted into psB16, respectively. shows. Figures 6 and 8 show psB301 plasmid DNA, respectively. A method for constructing DBHB13 plasmid DNA is shown. Figure 7 shows the synthetic NA BboA-1 and Bbgj! −
Figure 9 shows the sequence of the binding site between the α-amylase promoter region and the biotin synthase gene.

Claims (30)

[Claims] (1) DNA encoding genetic information involved in biotin biosynthesis reaction from desthiobiotin derived from Bacillus bacteria having biotin-producing ability. (2) The DNA according to claim 1, wherein the DNA encodes the amino acid sequence shown in FIG. 5 or an amino acid sequence having substantially the same function. (3) Claim 2 in which the DNA has a base sequence corresponding to positions 285 to 1277 in the base sequence shown in FIG. 5 or a base sequence having substantially the same function as the base sequence. DNA described in section. (4) A patent claim in which the DNA encodes the amino acid sequence shown in Figure 5 or an amino acid sequence having substantially the same function as that, and has a translation initiation codon upstream thereof and a translation termination codon downstream thereof The DNA according to the range item 1. (5) The DNA encodes the amino acid sequence shown in Figure 5 or an amino acid sequence having substantially the same function as that, and has a Shein-Dalgarno sequence and a translation start codon upstream thereof, and a translation stop codon downstream thereof. The DNA according to claim 1. (6) The sheain-Dalgarno sequence is GAAAAGGGAG
The DNA according to claim 5, which is AATGT. (7) The DNA according to claim 5, wherein the DNA is a base sequence corresponding to positions 265 to 1280 in the base sequence shown in FIG. 5, or a base sequence having substantially the same function as the base sequence. DNA. (8) A recombinant vector incorporating DNA encoding genetic information involved in biotin biosynthesis reaction from desthiobiotin derived from Bacillus bacteria capable of producing biotin.
. (9) The recombinant vector according to claim 8, wherein the DNA encodes the amino acid sequence shown in FIG. 5 or an amino acid sequence having substantially the same function. (10) The DNA is at position 285 in the base sequence shown in Figure 5.
10. The recombinant vector according to claim 9, which has a base sequence corresponding to positions 1 to 1277 or a base sequence having substantially the same function as the base sequence. (11) The recombinant vector according to claim 8, wherein the DNA is integrated under the control of a promoter and in close proximity thereto. (12) The recombinant vector according to claim 11, wherein the promoter functions in bacteria of the genus Escherichia, Bacillus, or Pseudomonas. (13) The recombinant vector according to claim 8, wherein the vector is a plasmid. (14) A bacterium transformed or transduced with a recombinant vector incorporating DNA encoding genetic information involved in biotin biosynthesis reaction from desthiobiotin derived from a Bacillus bacterium capable of producing biotin. (15) The bacterium according to claim 14, wherein the recombinant vector incorporates the DNA under the control of a promoter and in close proximity thereto. (16) Claim 14, wherein the bacteria belong to the genus Escherichia, Bacillus, or Pseudomonas.
Bacteria listed in section. (17) The bacterium according to claim 16, wherein the bacterium has the ability to produce desthiobiotin. (18) The bacterium according to claim 15, wherein the bacterium belongs to the genus Escherichia, and the promoter functions in bacteria belonging to the same genus. (19) The bacterium according to claim 15, wherein the bacterium belongs to the genus Bacillus, and the promoter functions in bacteria belonging to the same genus. (20) The bacterium according to claim 14, wherein the DNA encodes the amino acid sequence shown in FIG. 5 or an amino acid sequence having substantially the same function. (21) The DNA is at position 285 in the base sequence shown in Figure 5.
21. The bacterium according to claim 20, which has a base sequence corresponding to positions 1 to 1277 or functions substantially the same as the base sequence. (22) The bacterium according to claim 14, wherein the recombinant vector is a hybrid plasmid. (23) A bacterium that is transformed or transduced with a recombinant vector incorporating DNA encoding genetic information involved in biotin biosynthesis reaction from desthiobiotin derived from a Bacillus bacterium capable of producing biotin. A method for producing biotin, which comprises culturing in a medium containing a suitable carbon source in the presence of a synthetic precursor to produce biotin, and separating and purifying the produced biotin. (24) The recombinant vector is under the control of a promoter,
24. The method according to claim 23, wherein said DNA is incorporated in close proximity thereto. (25) Claim 2 in which the bacterium belongs to the genus Escherichia, Bacillus, or Pseudomonas.
The method described in Section 3. (26) The method according to claim 23, wherein the precursor is desthiobiotin. (27) Claim 2, wherein the precursor is vimelic acid or azelaic acid, and the transformed or transduced bacterium has the ability to produce desthiobiotin.
The method described in Section 3. (28) The recombinant vector is under the control of a promoter,
28. The method according to claim 27, wherein said DNA is incorporated in close proximity thereto. (29) The method according to claim 27, wherein the bacteria having the ability to produce desthiobiotin is a bacterium belonging to the genus Bacillus. (30) The method according to claim 29, wherein the bacterium belonging to the genus Bacillus is a strain belonging to the genus Bacillus sphaericus.