JPS63173591A - Dna sequence encoding biotin biosynthetic enzyme, vector containing the same, cell transformed by said vector and production of biotin using said cell - Google Patents

Abstract

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

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to the production of biotin by fermentation using recombinant DNA technology.

(Prior Art and Problems to be Solved by the Invention) Biotin is one of the vitamins required by humans, animals, plants, and certain microorganisms.

Biotin is isolated from egg yolks and can also be found in brewer's yeast, grains,
It is found in various organs in free form or protein-bound form.

It has been proposed to use this vitamin in particular as a regulator of skin metabolism, inter alia in the treatment of IIl&rhoea dermatitis in humans.

Although various other sources are known in the art, biotin is derived from certain microorganisms, particularly Bacillus sphaericus (Bacillus sphaericus).
It is synthesized by Bacillus species such as Bacillus 5phaericus).

FIG. 1 shows the biosynthetic chain of biotin using pimelic acid as a raw material in this type of microorganism. This chain contains steps using five different enzymes, and the genes used in these are, in order, bio C, bio
They are called F, blo A, blo D and blo B.

Systematic studies of biotin production by industrial fermentation from pimelic acid have shown that certain microorganisms, particularly those belonging to the Bacillus sphaericus species, overproduce vitamin intermediates (vltaiers) as well as biotin. became clear. (As used herein, "vitamin intermediate" refers to various intermediates that produce biotin.) Regarding these bacteria, among the biotin precursors, D
TB (desthiobiotin) ultimately becomes the dominant component produced in much larger amounts than the molecule biotin.

There are strong transcriptional controls that suppress biotin synthesis depending on the amount of biotin present in the biotin.

This suppression occurs similarly in E. colt and Bacillus sphaericus.

However, feedback inhibition does not regulate biotin biosynthesis in E. coli, and this type of regulation is unknown in Bacillus sphaericus.

To fully elucidate the production of extremely large amounts of DTB from pimelic acid (relative to small amounts of biotin) in Bacillus sphaericus, the mechanism and regulation of the biotin biosynthesis pathway in this bacterium will require considerable research. Further molecular biological studies are needed.

In an initial study of several extracted semi-purified biosynthetic enzymes from B. sphaericus strains that produce DTB, E.
No significant difference was observed between the biotin enzyme and the known biotin enzyme in E. coli.

However, if the yield of biotin is improved,
Such a strain of Bacillus sphaericus (11'0352
5. Production of biotin by industrial fermentation of MCl89370) would be competitive with existing chemical methods. To achieve this aim, it is advantageous if in these strains an essential hyperexpression of all biosynthetic genes for biotin is obtained.

The selection of derepressed E. coli strains by resistance to biotin analogues such as α-dehydrobiotin or selection of mutant strains that secrete excessive biotin is of course known. Also known are cis-dominant mutations that affect the synthesis of all biotin genes organized in a bipolar operon in a pleiotropic manner. Trans-acting mutations are also known, but in addition to being able to abolish transcriptional control of the biotin gene, these often have pleiotropic properties that can affect the general physiology of the cell.

These traditional microbiological selection methods are suitable for Bacillus sphaericus, given that the molecular control of biotin biosynthesis and the general organization of the biotin gene are similar in Bacillus sphaericus as in E. coli. It can also be applied to Sphericus strains.

In addition to these assumptions, the use of mutant strains in large-scale industrial fermentations and continuous cultures requires the selection of mutants in which reversion is extremely unlikely, and in Bacillus sphaericus This is a difficult task, as no highly accurate genetic analysis methods currently exist.

Another method consists of cloning all genes involved in the biotin biosynthetic chain from a DTB-producing strain of Bacillus sphaericus and then modifying the 5' regulatory region in vitro so that it is no longer transcriptionally regulated. .

Furthermore, by cloning and manipulating these genes in vitro, using known genetic engineering techniques, especially those involving the use of strong promoters that are functional for Bacillus sphaericus strains, we can improve the ability of these common tissues. Studies will enable and improve these in vivo transcriptions.

All these hyperexpressed genes that are no longer regulated by biotin (but can be regulated inducibly, e.g. by heating or introduction of inexpensive substrates) are then transformed into Bacillus cells using known transformation, translation or conjugation-fluidization techniques. can be reintroduced into the bulk of Sphaericus.

In addition, these genes can be used in the food industry to
Different microorganisms are permeable to pimelic acid, such as Bacillus subtilis.
btllis), Saccharomyces cerevisiae (Sac
charotayccs crcvislac) or pseudomonas (Pscudomonas).

Furthermore, fixation of this genetic information in microorganisms can be achieved by directional integration in the chromosome or by self-selection of plasmids, as described for example in FR 84712598.

The cloning and characterization of the bio B gene of Bacillus sphaericus has already been published in Japanese Patent Application Laid-Open No. 1985-18 (1985)-18 (
i992, No. 61 (1988)-66532 and No. 81 (198B)-95724.

(Means for Solving the Problems) In particular, the present invention is directed to
A gene, blo D gene, blo F gene.

It relates to a DNA sequence encoding an enzyme produced by one of the bio C gene and the bio H gene.

Some of these DNA sequences according to the invention are clustered together with the previously identified bio B, thus the whole group covers most of the biotin biosynthesis pathway.

Among the interesting DNA sequences, we should mention those encoding enzymes produced by the following genes: bio B and bio D bio B, bio D and bio Abio B
, blo D, blo A and bioF0 Using sequences of this type in appropriate vectors makes it possible to produce biotin from various vitamin intermediates.

As described below, biotin is produced by the following gene: bjo F
and blo C bio F and blo H bio F, and DNA sequences encoding enzymes produced by blo C and blo H, and in addition to these sequences, the following genes: blo A bio A and bioD, or bio A, It can be produced by fermentation using vectors expressing sequences encoding the products of bio B and bio D, respectively.

Although the DNA sequences mentioned above can be of various origins, it is preferred to use sequences derived from Bacillus strains, especially Bacillus sphaericus strains.

As mentioned above, these DNA sequences are subject to the control of selected elements provided for their efficient transcription in the host strain, in order to stop the natural regulation by biotin and place these DNA sequences under the control of selected elements that provide for their efficient transcription in the host strain. It is particularly preferred that the natural sequence capable of regulating the transcription of enzymes involved in the biotin biosynthesis pathway in the protobacterium has been lost.

The invention particularly relates to all or part of the sequences shown in Figures 4-8 and 17-19 and encoding one of the genes mentioned above.

The DNA sequences of the invention can be used in a variety of ways.

The DNA sequence in question is preferably carried in a plasmid vector which allows transformation of bacteria and contains all the elements for the expression of the corresponding gene.

This plasmid can be of an autonomous self-replicating type as described above, or can be of a form that integrates into the chromosome of the host strain.

Various techniques used for this purpose are known or described in the Examples.

In the case of an integrating vector, it should contain at least one sequence homologous to a sequence present in the genome of the strain to be transformed, thereby ensuring chromosomal integration. It is clear that both homologous sequences corresponding to natural genomic sequences and homologous sequences introduced by other plasmid integrating vectors can be used.

If the vector is autonomously replicating, it will contain an effective source of replication in the host cell.

Similarly, these various plasmid vectors should contain selection elements such as genes for resistance to antibiotics and/or marker genes under the control of the promoter of the strain to be transformed. Can be done.

Among the cells that can be used as host strains, mention should be made of bacteria, in particular of the genera Esherlichja, Bacillus and Pseudomonas, and yeasts, in particular of the genus Satucharomyces.

Particularly preferred host cells include Bacillus sufyuricus,
Bacillus subtilis and Bacillus subtilis should be mentioned.

Particularly preferred strains transformed by the DNA sequences of the invention are those that contain other genes involved in this biosynthetic pathway, namely F, A, as described herein.

It has already been transformed with a vector for expressing the D and B genes.

Under these conditions, the introduction of the bio C and bio H genes into a bacterium enables the bacterium to synthesize biotin from the first vitamin intermediate of the biosynthetic pathway, i.e., pimelic acid, thereby making it possible to synthesize biotin on an industrial scale. considerable economic benefits can be obtained.

In the present invention, the plasmid pTG475 can be particularly used as a plasmid integration vector, and this vector will be described later, and it contains an inducible promoter.

If the vector contains an autonomously replicating source, the DNA sequences encoding the enzymes mentioned above are preferably flanked by control elements ensuring their expression in the host strain.

They have a strong promoter, especially at the 5' end, which is effective throughout the body, and in some cases, a termination sequence is used, for example when the host strain is yeast or other microorganisms that require a termination sequence. Contains other elements such as

The present invention also relates to cells transformed with the vector plasmids of the present invention. Among these cells, mention should especially be made of bacteria, especially of the genus Bacillus, Escherichia or Pseudomonas, and yeasts, especially of the genus Satucharomyces.

Among the strains that can be transformed with these vectors, mention should be made of those that already produce biotin or one of its vitamin intermediates.

Furthermore, the present invention comprises fermenting a propagation substrate containing at least one of the vitamin intermediates of pimelic acid or biotin by cells permeable to biotin or said vitamin intermediate as described above, and recovering the biotin produced. The present invention relates to a method for producing biotin.

The method of the invention can be modified in many ways.

In particular, vitamin intermediates can be directly produced using cells transformed with the vector of the present invention. especially,
Transformed strains containing the blo F, blo H and bio C genes are capable of producing KAPA from pimelic acid, and this conversion of KAPA to biotin can be achieved, for example, by strains carrying complementary genes A, B. be done.

The two fermentations can be carried out sequentially or co-fermentation can be carried out if the strains are compatible with each other.

It is also possible to envisage complementation of strains carrying only part of the gene in question, and it is also possible to transform strains that already produce biotin to over-productivity.

(Example) Hereinafter, the present invention will be described in detail with reference to the drawings.

Example 1 a) E. Bacillus sphaericus IPO3525 in 200d PA
B double land (DIPCOBacto antibiotic lease land 3.1
7.5 g/l) at 37° C. for 17 hours. Cell bacteria were collected by centrifugation, and then total DNA
using Saito's method (Salto et al, BBA
19B3.72.619-829) from the cells. In this way a pure NA of 450μ3 was obtained.

Total DNA 20μ3 was converted into old ndm (3U/u gDNA
) was completely restricted. pBR322 is tllndII
[After complete digestion, it was treated with alkaline phosphatase.

Genomic DNA digested with 111ndIII (2tt'j) and pBR3 treated as above (1 μg)
22 with 30mM NaC, 30a+M p)1
7,5 Trls-HCl.

Mg CQ of 10IIIM, z, p of 0.2s+M
Hg EDTA, 2 mM DTT, 0.5n
+M pH 7 ATP and 0.1/71ff/d B
Hybrid recombinant plasmids were obtained by mixing with 2 units of T4 ligase (Bochringcr Mannhcim) in reaction buffer containing SA.

Incubation was carried out at 14°C for 16 hours. Then, using the E. coli C600r, -mK+ strain, LB
A transformation test (Cohenat
al. (1972) PNAS 69.2110-21
An aliquot of this ligation mixture was added to 14).

Four different pools of plasmid DNA were then extracted, each with a 10'
This corresponded to several clones.

These DNA pools were then used to transform various old 0 mutants of E. coli, and transformants were incubated in the presence of ampicillin (100 μ3/d) on LB medium or in response to this antibiotic. Selection was made by prototrophy for biotin as well as resistance (LB medium + ampyridine 100 μ9/d + avidin 0.2 μ/
m).

The results obtained are shown in Table 1.

Table 1/μgDNA transformant/μgDNAC268
*ΔbioA, his > 1033 (pool NO, 4) C173*ΔbioD, his >
1032 (Pool NQ4)*: C1ary an
d Campbell (1972) J, Bacteri.
The 112,830° plasmid was isolated from ampicillin+avidin selected clones and analyzed using restriction enzymes. Three plasmids (2 from strain 0268 and 1 from C173) contain BamHI, 5a with one Bgln and two Sph I positions.
ll, PsLI.

EcoRV, without PvuII or AvaI4
, and contained a 3 kb l1indlIr insert.

The restriction map of one of these plasmids, PTG1400, is shown in FIG. By prototrophic selection for ampicillin resistance and biotin, C.
10g (Δbio A), C173 (ΔbjoD
), R877 (Δbio D19) (Clcary
and Campbcll, 1972) or C
The 6 strains of 162 (bio B) also correspond to the frequency obtained in the case of ampicillin alone selection (1 μJ of DNA
03) frequency could be transformed again with equal success. When using pTG1400, R
878 (bio C23) or R901 (Δbio
A-D) strain (Cleary and Can+pbel
1, 1972) could not be obtained.

b) The normal replicating form of Bacillus subtilis in Bacillus subtilis, as it is known that deletions can occur with extremely high frequency in foreign inserts cloned into plasmids. Complementation of the blo mutant was thought to be difficult. Therefore, we developed a new method based on complementation testing using non-replicating plasmids. If there is a homologous region between the genomic DNA and the plasmid, the integration of the non-replicating plasmid into the genomic DNA of Bacillus subtilis will occur at a fairly high frequency (approximately 104 transformants/μg
DNA).

The first step was to use the plasmid pTG, the structure of which is shown in Figure 3.
475 into different bio mutants of Bacillus subtilis.

Plasmid pTG475 contains the XylE gene encoding the enzyme C2,3-oxygenase (Zukowski et al.
L al, (1983) PNAS USA, 80.
1101-1105), which can be used as a colored (yellow) marker and is expressed under the control of the inducible promoter of the Bacillus subtilis levansucrase gene. Additionally, this plasmid has a CAT gene that confers chloramphenicol resistance. CAT and XylE genes are in pB
It was inserted into R322.

This plasmid was integrated into the chromosome of the following Bacillus subtilis strains: bioA strain: JK [33173 (bioA 173.
aro C932: C1l pal (1975) J, B
acLcriol, 121.1-8).

bior3 strain: 13GSCIA92 (bjoB 14
1. aro C932Sac A 321, Arg A
2+C1l Paj (1975) J, Bacterio.
l, 12+.

l-8゜biol12 strain: JKB 3112 (biol12;
C1l Pa1 (1975) J, Baeteriol,
121.1-8).

In this case, R, J. Boyland (1972
), J, Bactcrlol, 110.281-290
By competent cell transformation technology, selection is made of TB
AB (D l ['CO blood tryptose agar medium)
The test was carried out using a solution containing 3μ9/ml of chloramphenicol.

Various tests were performed on the transformed clones. These clones turned yellow when induced with sucrose and also showed blo A, bi.

In a Southern hybridization test for B and biol12 strains, integration of pTG475 into the promoter of the levansucrose gene by simple recombination was observed. These transformed strains of Bacillus subtilis were transformed into blo ATG 1. Referred to as blo BrO3 and blol12T G 3. Plasmid pTG4
Since 75 introduces the pBR322 sequence into the Bacillus subtilis genome, any foreign gene containing these similar sequences can be integrated by homologous recombination.

In the second step, Bacillus subtilis bio ATGI, bio BrO3 and old o
3 strains of 112T G were transformed. Select LB leased land+
Avidin 0.2μ/d + Chloramphus Nicole 10μ9
It was carried out on 7m1.

This plasmid contains bloATGl and bio87
It was found that it was possible to select at an extremely high frequency transformants that were prototrophic for biotin derived from the 02 strain but not for biol2T G3. Therefore, pTG1400 is biol1
2 Do not complement mutations.

c) Final characterization of the Hindu insert of pTG1400 Similar old n in the genomic DNA of Bacillus sphaericus IFO3525 corresponding to the insert of pTG1400
Southern hybridization tests were performed to detect the dIII fragment.

Under vigorous hybridization conditions (50% formamide, 0.6% Denhard L solution, 0.1%
Translation for in vitro polymerization (SDS, 3 x 5 SC, 42 °C) (2 °5 x IO)
Plasmid pTG14 labeled with 32p by radionucleotide incorporation (cpm/μg DNA)
Using 00, a single 4.3 kb HlndI[I band could be seen after 6 hours of autoradiography at -80°C. It was confirmed that pBR322 did not cross-react with the genomic DNA of Bacillus sphaericus IF03525 under these hybridization conditions. Under these similar conditions pT G
1400 as a probe, any positive reaction was caused by the old ndIII of Bacillus subtilis BGSCIA289.
It was not detected in the treated genomic DNA or in the E. coli C600 IIIndnl treated genomic DNA.

The shotgun method, which is the systematic re-loning of fragments obtained by sonication, 'cyclone deletion (c
cyclone dcletion) J or oligonucleotide primer extension to 4°3 kb.
The entire DNA sequence of the old mouth dm fragment was analyzed.

In the case of the shotgun method, plasmid pTG1400 was degraded by sonication. After treating the DNA segment with phage T, DNA polymerase, the plant-ended fragments were mixed with a low melting point agarose gel to separate fragments approximately aoobp in size.

Cloning of these fragments was carried out using 51llaI
The procedure was carried out in M13 vector which had been digested with phosphatase.

Clear plaques that did not cross-hybridize with pBR322 were screened and 100 clones were sequenced. The results were analyzed by computer.

When sequencing DNA, a series of overlapping clones (
Recent methods of generating cyclone systems (1?, M,
K, Dale, B, A, McClure,
J, P, l1ouchins (1985) P
I asm 1d 13.3l-40) was used. This method was performed in parallel with the shotgun method to confirm the results.

Furthermore, by this method, it is possible to
A limited deletion plasmid containing the b io gene was obtained.

The complete sequence of the ll1ndIn fragment of pTG1400 is shown in Figures 4.5, 6.7 and 8.

According to this sequence computer analysis, the fragment consists of 4
It was revealed that the protein has the ability to encode two long open reading frames (LORP).
gaho) region is underlined. Palindromic regions are underlined at the 5' end of sequences that may represent translation termination surfaces.

Detailed complementation analysis shows that the first LORr' region (with three predicted translation initiation values) (Fig. 5) is bi
o It was revealed that it corresponds to the D gene.

E. coli strain C3R803 (rec^l, phr-1, u
vrA6. Lhr-1゜Ieu-6, thi-1, ar
gE3, lacYl, galK2. ara14
, xyl15 , atIf, proA2,5Lr-3
1, tsx-33, 5upE44. An experiment known as [n+axicells J] was carried out on F-, λ-), and it was revealed that this region encodes a polypeptide with an apparent molecular weight of about 25 kd.

a second LOR (with 4 expected translation start values)
The P region (Figure 6) corresponds to the bio A gene.

"Maxi-cell" experiments using E. coli C3R603 revealed a polypeptide with an apparent molecular weight of approximately 40 kd corresponding to the product of the bio A gene.

It was not possible to determine the function of the third LORF sequence, designated the Y gene (Figure 7). In view of the extremely high hydrophobicity and the presence of predicted polymorphic sequences in the coding region, we believe that this LORF encodes a protein that interacts with membranes when transcribed and translated. Since this Y gene forms a cluster with other 10 genes (A, D, and B), it is thought that it encodes another function involved in biotin metabolism.

4th LOR (with 3 expected translation start values)
The F region (Figure 8) has already been identified and is the region that coats the b1oB gene. Here, this gene or bi
It was shown to be connected to the o A and bio D genes in a cluster.

Complementation analysis using a plasmid containing the subcloned region of the 4.3 kb ll1ndI[I insert in pTG1400 revealed that the first LORF region corresponds to the product of the D gene, as shown in Figure 9. However, it became clear that the second LORP region corresponds to the product of the A gene.

Example 2 Following the methodology described above, Bacillus sphaericus I as described above was used to transform the bioF12 mutant of E. coli.
The Illndm genomic DNA library of FO3525 was used.

The results obtained are shown in Table 2.

Table 2/μ9 DNA transformant/μ9 DNAR8
74*bioF 12. his >10'
2 (Pool No, l) (Each pool) 2 (Boolean Nα3) 4 (Boolean Nα4) *: C1early and Campbell (19
72) J, Bacterol, 112.830 Plasmids were isolated from selected clones on loans containing ampicillin and avidin and analyzed using restriction enzymes.

Two of these plasmids are Spl, KpnI.

Hpal, Ndcl, Aval, C1al, BglI,
Xmn1, PvuII, BglII, Xbal, 51Ilal, with 5caI and 5tu1 positions
Pstl, 5ail, Nrul.

Baa+HI, PvuI, EcoRI, Hin
d II, EcoRV, Fspl, Apal, B
Approximately 4.5 without a1l or AatII position, etc.
kb and two 11[ndIII inserts of approximately 0.6 kb.

By using one of these plasmids, pTGL481, and selecting for ampicillin resistance and prototrophy to biotin, E. coli R87
4 strains (bioP12.his) were treated with ampicillin (to
It was possible to transform with an average frequency corresponding to the frequency obtained by selection with >& /μg DNA).

According to subcloning experiments, the 4.5kb old nd■
The only fragment is E. coli bi of strain R874.

It was found to encode a KAPA synthetase function that ensures complementation of the F12 mutant.

Southern experiments were performed to detect the 4°5 kblind m fragment in the genomic DNA of Bacillus sphaericus lPO3525 in comparison to the 4.5 kb insert of pTG1418. Extremely aggressive hybridization conditions (50% formamide, 3XSSC1
2X with M2B5 G 1425 (translation for in vitro polymerization) using 0.1% SDS, 0.6% Denhardt solution, 42 °C).
4,5 kbHi labeled with 32p by incorporating radionucleotides at 10' cpm/μg DNA
When using phage M13) containing nd m, -80
After 7 hours of autography at °C, approximately 4.5
A former ndnI band of kb was observed.

Under similar conditions, Bacillus subtilis BGSCIA2
89 l1jndI [E also in genomic DNA treated with I.

Genomic DNA of coli C600 treated with IIIndIII
No positive reactions were observed.

Using the 8.3 kb S ph I fragment of pTG140B, which overlaps the 3' end of pTG1400, p
8 of p5BOI, which overlaps with the 5' end of TG1400.
Even with the 2 kb Mbol fragment, pTG14
No cross-reactivity was detected between the 00 insert and the pTG1418 insert (see 10).

We analyzed the restriction map of the insertion site of pTG1418 and
Shown in the figure.

Complementation experiments and Southern analysis revealed that the bio F gene of Bacillus sphaericus 1FO3525 was not linked to the bio A, bio D, and bio B genes of the same microorganism.

Complementation of Example 3 B, B. subtilis mutant blol12 was tested in a nutritional test (C, Il, Pa 1.1975.J, Bacteri.
ol 121.1-8). In this mutant, pcellasmid p
Incorporating TG475 at the sac R-saeB locus,
The resulting new strain was named biol2T G3.

Various plasmids (p TG1418.1420,14
22.1435.1436 and 1437, Figure 13~
Transformation of the biol2T G 3 strain was performed using the method shown in FIG. 16) followed by selection on LB medium + ioμg/d of chloramphenicol + 500 μg/day of avidin. The results of complementation are shown in Table 3. The B. subtilis mutant blol12 likely arose in the bio F gene, since similar results were obtained in a cross-over study with the E. coli mutant R874 (blo F, his).

Analyzing the resulting complementarity with respect to the plasmid used, it is possible to localize the blo F gene on the insert of pTG1418 to the position of the fragment flanked by the Xmn1 and Ncol restriction sites (21st (See Fig. 23).

Table 3 If I nd m of p TG1418 5.1 kb
Insertion part 10+p TG1422 bi
o 3.1 kb of C containing P and part of LORFν
la I insert 10 + 2 kb C1a containing part of pTG1420X and LORP W(7)
l-11ind m insertion part
-pTG1436 1.3kb Xmn I-Ncol insert containing bio F gene gene 10+p TG14
37 bio Neo l of LJkb containing F gene
-Xll1nl insertion section 10pTG143
5 1. OI? 0.6 kb (7) 11 containing I'
pa I-Pvun insertion part - Example 4 11indlII insertion part M! 3T G 131 at the corresponding color positions of the polylinker.

The so-called [cyclone] method was applied to this plasmid M137.
G 1425 and the results were analyzed by computer. Using certain oligonucleotides, sequencing revealed some reading differences between the two complementary strands.

The arrangement is shown in detail in FIGS. 17 to 19. The inserts have molecular weights of 18,462 Daltons and 2,804 Daltons, respectively.
It is recognized that it contains three open reading frames encoding proteins (subunits) of 8 Daltons and 4294 (1 Daltons).The last gene along the reading direction is bi
o located in the region identified as F, and this B,
The molecular weight of the Sphaericus protein is that of E5 coli KAPA synthetase (M, A, l Eisenberg, 19
73, adv, Enzymol, 38.317-37
It was of the same order of magnitude as 2).

The juxtaposition of these three open reading frames is plasmid pTG 14
Like the case of 00, this would be typical of an operon structure. Upstream of the first gene, plasmid pT
It should be noted that a 15 base pair sequence (underlined in Figure 17) that is also present upstream of the first gene (bioD) of the G1400 insert can be identified. This significant feature may suggest that the two groups of B. sphaericus biotin genes are subject to at least one common regulation. This regulation is defined as previously described for B. sphaericus KAPA synthetase (bioF) (Y.
i, ni, 5ato, Y, Tan1 and K, O
gata, 1973. Agric, Biol, Chem
J7. 1335), which can be regulated by biotin (or its derivatives).

On the 3' side of the last gene sequenced it is possible to identify a sequence (underlined in Figure 18) having the characteristics of a transcription terminator.

Examples Plasmids pTG 1418 (1433) and p
Traditional complementation assays for E. coli mutants were performed using the TG1440 plasmid, pTG1418 and its derivatives.

E, coli mutant R87g (bio C, his
) were transformed with plasmids pTG1418 and pTG 1433 (see Table 4) and then treated with ampicillin ± 200μ of LB medium 10 IOD μ9/d.
When spread on avidin of /immune, growth was observed after 36 hours of incubation at 37°C. The frequency of transformed clones on this plot was of the same order as that determined on the LB plot plus 100 μg/d ampicillin.

Table 4 p TG1418 103 103 little p TG1
433 103to3 weak p BR3221030 (38 h after incubation at 37°C) Growth of transformed clones normal in LB + ampicillin in the absence of biotin (LB + ampicillin + avidin; no biotin, minimal oxidation (added acid). Mutant R878b
This complementarity of the biotin auxotrophy of ioC is in light of the fact that similar mutants show no residual growth on borrows lacking biotin when transformed with various plasmids derived from pBR322. , has great meaning.

The two inserts of plasmids pTG1400 and pTG1418 were cloned into pBR322 to create plasmid pTG1400 and pTG1418.
TG1440 (Figure 20) was obtained. This plasmid p
TG1440 is E, Cori's C261 (Δbio FCD
, hiS), 100 μg/L of LB double medium
Allow selection of clones on m1 ampicillin + 200μ/mm avidin. The frequency of transformation obtained was directly comparable to that determined by 7TIJ on LB+ampicillin. Again, growth of these recombinant clones normal on LB double ampicillin was retarded in the absence of biotin. From these two results (complementation of the biotin auxotrophy of mutants bioC and bjoΔFCD), plasmid pT01
It was revealed that the insertion site of 418 also contains the bio C gene of B9 sphaericus. pTG1418
Various subcloned bodies (21st
Figures to Figure 23) are E.

It was not possible to obtain complementation in the E. coli bio C mutant. Only inserts with all three genes conferred effective complementation to the E. coli bio C mutation.

The mutant in the example was originally described as bio B (D, l1atrield, M, II of'nung
and M, 5chvartz.

1909, J, l3acterio! , 98';',
559-507). It was then determined that it does not secrete any vitamin intermediates and is capable of growing on inorganic substrates in the presence of KAPA, DAPA, 1) T13 or biotin. Next, Eisenbarb (Einc
nbcrg; 1985. 8nnals New Year
kAcademy of' 5ciencCs 447
, 335-349), this gene is pimeloin-CoA.
It was proposed that it encodes a subunit of synthetase (bioH). E. coli mutants that overproduce biotin (selected for their levels of vitamin secretion or α-dehydrobiotin resistance that enable the growth of bioB auxotrophs of E. coli) are all genetically It should be noted that it was identified as being located at the bioR locus. This locus is (bloAB
It encodes a multifunctional protein that is a repressor of the synthesis of the FCD operon to metsenger RN, and at the same time is a holoenzyme synthetase that binds biotin to the lysine residue of various apoenzymes with carboxylase function.

All biotin-overproducing E. coli mutants identified to date have been localized in the gene encoding the trans-active repressor, bt.

Since it is not among the operators of ABCD operon,
It is likely that other genes involved in biotin biosynthesis are subject to this regulation. According to the literature, the bio H gene is the most likely.

The insert of pT01418 is B, Sphaericus bt.

Since it contains the F and bio C genes, we investigated whether a third gene corresponds to the bjo H gene.

Complementation of the E. coli bio H mutant was effectively obtained by using plasmid pTGI433. The multiplication obtained on this leased land was here also delayed, but
It was fully significant when compared to the control. (See Table 5).

Table 5 p BR3221040 p TG1433, 103 a little less than 103 (36h
, after incubation at 37° C.) Necessary and sufficient conditions for the complementation of the bio H mutant are introduced into the body as in the case of the complementation of the bio C mutant l) TG1
The three genes of the 418 insertion are present at the same time (Figures 21-23).

bioC and bi in the absence of biotin, similar to the blo F mutant of E. coli and B. subtilis that can be complemented by a single gene of B. sphaericus.
o The growth of the H mutant is caused by l1ind of pTG141g.
was obtained only when a recombinant plasmid carrying the three genes of the III insert was introduced.

This is especially true for B. sphaericus bimeroy-CoA
Differences in enzymatic properties between the synthetase and the corresponding enzyme in E. coli (differences in affinity for substrates, especially for multienzyme systems) or limiting the metabolic flux of pimelic acid to KAPA in E. coli and B. , is thought to lead to a reduction in the synthesis of pimeloyl-CoA synthetase in S. sphaericus.

Example 7 Functional test of bioFCH gene B, 4 to 5 kb Ili d isolated from Sphaericus
E. coli bio F, bio H using a recombinant plasmid carrying an insert derived from the m fragment.
and bio C mutant complementation tests were carried out using this DNA
bio F, bio H and blo C above
It can be thought of as proving the existence of genes.

However, it is useful to supplement this information by testing the activity of the products of these genes cloned from B. sphaericus. E.

The product of the coli bio F gene is pimeloin-C.

(1'EIsenberg, 196g) o The products of the two genes blo C and bio H have not been clearly identified to date, but have been described in the literature. The proposed hypothesis (Elsenberg, 1985) suggests that these encode proteins involved in the biosynthesis of bimeroy-CoA. Tests were conducted to determine whether the bio F, C, H sequence specifically accomplishes the conversion of pimelic acid to KAPA.

FC of B. sphaericus due to significant expression levels of proteins related to F, C and H genes.
Insert carrying H-coding region (ligated pTG
EcoRV-3phI fragment of 1434 and pTG
143B (recovered in the combined form of 5phl-8phI fragments) was fused to the promoter of the gene conferring tetracycline resistance to pBR322 to obtain plasmid pTG1446.

This plasmid was then introduced into competent cells of the E. coli strain H. Next, this recombinant strain was used at 100μ9/rrd! of ampicillin and 0.5μ
20 g of glycerol, 30 g of proteose pebtone, 5 g of vitamin-free casamino acids, 1 g of 2HPO, 0.59 KC look.

0.5g Mg So, -7H2O,0, old G FeS
0.001 g M of O4-7H20, pH 6,8-7
nSO4・4Hz o, 20μ9 Thiamine-HC9J
The cells were cultured at 37° C. for 48 hours.

An aliquot of the supernatant (5 μ9 J) was then placed on a silica plate and subjected to phase separation analysis to separate the produced vitamin intermediates and biotin (chromatographic solvent:
n-butanol (60)/acetic acid (15)/water (25)
, vol/vol, solvent transfer: 10 cm>.

After specific transfer for each vitamin intermediate, the Satucharomyces cerevisiae collection strain, namely ATCC7754
KAPA was quantified using a biological assay.

Under these conditions, measurements of 85 μ9 KAPA (expressed as biotin equivalent) per d of borrowed supernatant were reproducible.

The controls used in this experiment were grown under the same conditions,
It was the same strain containing plasmid pBR322. No significant detection of KAPA was observed in this case.

Therefore, it has now become clear that the pTG1418 insert encodes the necessary and sufficient functions for the conversion of pimelic acid to KAPA, ie, the products of the old oC, H and F genes.

Deposit of strains representative of the invention E, coli strains CGOop TG 1400 and R87
4p TG1418 was collected on September 26, 1986 at the French National Collection of Microorganisms at the Institut Pasteur (la Co11ec).
tion Nationale dc Culture
s da Microorganismes de I
'In5titutPasteur, 28 rue
du Docteur-Roux 75724 Par
isCedcx 15) with deposit numbers 1-608 and l
Deposited under -609.

References P, P, C1early and A, Can+pbe1
1 (1972) J, BacLerol.

112.830-839゜M, M, Zukovsk i, D, F, Gaf
f'ney, D, 5peck, M, Ka
urf'mann, A, Findeli, A, enemy1se
cup and J, P, Lecocq (1983) P
NAS USA 80, 1101-1105°C, Il
, Pai (1975) J, Bacterol, 1
21.18, I? , J. Boyland, N. H. Me.
ndelson, D., l3rooks and P.E.
, Young (1972) J, Bactcriol, 1
10.281-290°R, M, Dalc, B, A
, Mc C1ure and J.P., Iouehi.
ns(198f3)PlaIlid 13.31-40
゜S, N, Cohcn, A, C, Y, Cheng an
d L, Hsu (1972) PNAS USA 69,
211.0-2114°Il, 5aiLo and K, 1. Miura (19
83) BBA 72,819-829゜M, A, Eis
enberg (1985) Regula LIon ol
' the blotinoperon, ^nnals
New York Academy or 5cie
nces.

447.335-349゜M, A, Eisenberg and C, Star (
196g) J, Bacteriol.

96.1291-1297°

[Brief explanation of the drawing]

Figure 1 is a process diagram showing the biotin biosynthesis pathway, Figure 2 is a schematic diagram showing the structure of plasmid pTG1400, Figure 3 is a schematic diagram showing the structure of plasmid pTG475, and Figure 4 is a schematic diagram showing the structure of plasmid pTG475. LO1? A schematic diagram showing the non-coding sequence upstream of the P sequence, Figure 5 is the first LOR corresponding to the blo D gene.
A schematic diagram showing the P sequence, FIG. 6 shows the second 1. A schematic diagram showing the ORF sequence, FIG. 7, is the third 1. of the above fragment corresponding to the Y gene. FIG. 8 is a schematic diagram showing the fourth LORP sequence of the above fragment corresponding to the bio B gene. FIG. 9 is a schematic diagram showing the complementation test of pTG1400.
Figure O is a schematic diagram showing the complementarity test between different plasmids. Figure 11 is a schematic diagram showing the structure of plasmid pTG141g. Figure 12 is a schematic diagram 1 showing the complementation test of pT01418.
, Figure 13 is a restriction map of the B. sphaericus insert used in the plasmids, Figure 14 is the restriction map of the plasmids pTG1418 and pTG14.
Figure 15 is a schematic diagram showing the structure of plasmids pTG1422 and pTG14.
Figure 16 is a schematic diagram showing the structure of plasmids pTG1436 and pTG14.
Figure 17 is a schematic diagram showing the structure of LORF
'Schematic diagram showing P sequence, Figure 20 is plasmid pTG
Schematic diagram showing 1440, Figure 21 shows plasmid pTG1
Restriction map of the insertion site of 418, Figures 22 and 23 are pT used for complementation test.
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Claims (1)

Claims: (1) A DNA sequence encoding an enzyme produced by one of the bioA, bioD, bioF, bioC, and bioH genes involved in the bacterial biotin biosynthesis chain. (2) The DNA sequence according to claim 1, which encodes an enzyme produced by the bioB and bioD genes. (3) The DNA sequence according to claim 1, which encodes an enzyme produced by the bioD, bioA, and bioB genes. (4) The DNA sequence according to claim 1, which encodes an enzyme produced by the bioD, bioA, bioB, and bioF genes. (5) D according to any one of claims 1 to 4, which has lost the natural sequence that controls transcription of enzymes in the biotin biosynthesis pathway in protobacteria.
NA sequence. (6) The DNA sequence according to any one of claims 1 to 5, which is derived from a Bacillus strain. (7) The DNA sequence according to claim 6, which is derived from a Bacillus sphaericus strain. (8) The following gene groups involved in the bacterial biotin biosynthesis chain: bioF and bioC bioF and bioH DN encoding an enzyme produced by one of bioF, bioC and bioH
A array. (9) In addition to the enzyme, the enzyme encodes at least one enzyme produced by one of the following gene groups: bioA, bioA, and bioD. DNA sequence. (10) The DN according to claim 8 or 9, which encodes an enzyme produced by the bioD, bioA, and bioB genes.
A array. (11) The DNA sequence according to any one of claims 8 to 10, characterized in that it has lost a natural sequence that controls the transcription of enzymes in the biotin biosynthetic pathway in protobacteria. . (12) The DN according to any one of claims 8 to 11, which is derived from a Bacillus strain.
A array. (13) The DNA sequence according to claim 12, which is derived from a Bacillus sphaericus strain. (14) A plasmid vector containing at least one DNA sequence encoding an enzyme produced by one of the bioA gene, bioD gene, bioF gene, bioC gene, and bioH gene involved in the bacterial biotin biosynthetic chain. . (15) The vector according to claim 14, characterized in that it contains a DNA sequence that enables integration of the vector into the chromosome of a host cell. (18) The vector according to claim 15, characterized in that it contains at least one sequence homologous to a sequence that is present in the genome of the strain to be transformed and allows integration. (17) The vector according to claim 16, wherein the homologous sequence is a natural genome sequence. (18) The vector according to claim 16, wherein the homologous sequence is a sequence integrated into the genome by a plasmid integrating vector. (19) The vector according to claim 18, wherein the plasmid integration vector contains at least one antibiotic resistance vector and a marker gene under the control of the promoter of the strain to be transformed. . (20) The vector according to claim 19, wherein the promoter is an inducible promoter. (21) The plasmid integration vector is plasmid p
Claim 2 characterized in that it is TG475.
Vector described in item 0. (22) The vector according to claim 14, which contains an autonomous replication source. (23) The vector according to claim 22, wherein the autonomous replication source is an autonomous replication source in a Bacillus strain or an Escherichia strain. (24) Claim 22 or 23, characterized in that the DNA sequence encoding an enzyme involved in the biotin biosynthesis chain is sandwiched between elements for ensuring expression in the host cell. Vectors listed. (25) The vector according to claim 24, wherein a promoter and a terminator that are effective in the host strain are present in the elements. (26) The following gene groups involved in the bacterial biotin biosynthesis chain: bioF and bioC bioF and bioH DN encoding an enzyme produced by one of bioF, bioC and bioH
A plasmid vector containing at least one A sequence. (27) The vector according to claim 26, characterized in that it contains a DNA sequence that enables integration of the vector into the host cell chromosome. (28) The vector according to claim 27, characterized in that it contains at least one sequence homologous to a sequence that is present in the genome of the strain to be transformed and allows integration. (29) The vector according to claim 28, wherein the homologous sequence is a natural genome sequence. (30) The vector according to claim 28, wherein the homologous sequence is a sequence integrated into the genome by a plasmid integrating vector. (31) The vector according to claim 30, wherein the plasmid integration vector contains at least one antibiotic resistance vector and a marker gene under the control of the promoter of the strain to be transformed. . (32) The vector according to claim 31, wherein the promoter is an inducible promoter. (33) The plasmid integration vector is plasmid p
Claim 3 characterized in that it is TG475.
The vector described in Section 2. (34) The vector according to claim 26, which contains an autonomous replication source. (35) The vector according to claim 34, wherein the autonomous replication source is an autonomous replication source in a Bacillus strain or an Escherichia strain. (36) Claim 34 or 35, characterized in that the DNA sequence encoding an enzyme involved in the biotin biosynthetic chain is sandwiched between elements for ensuring expression in the host cell. Vectors listed. (37) The vector according to claim 36, wherein a promoter and a terminator that are effective in the host strain are present in the elements. (38) A plasmid vector containing at least one DNA sequence encoding an enzyme produced by one of the bioA gene, bioD gene, bioF gene, bioC gene, and bioH gene involved in the bacterial biotin biosynthetic chain. cells transformed by. (39) The cell according to claim 38, which is a bacterium selected from the group consisting of Bacillus, Escherichia, and Pseudomonas. (40) The cell according to claim 38, which is a yeast of the genus Saccharomyces. (41) The cell according to claim 39, wherein the bacterium is selected from the group consisting of Bacillus sphaericus, Bacillus subtilis, and Escherichia coli. (42) The cell according to any one of claims 38 to 41, which produces biotin or its vitamin intermediate. (43) The following gene groups involved in the bacterial biotin biosynthesis chain: bioF and bioC bioF and bioH DN encoding an enzyme produced by one of bioF, bioC and bioH
A cell transformed with a plasmid vector containing at least one A sequence. (44) The cell according to claim 43, which is a bacterium selected from the group consisting of Bacillus, Escherichia, and Pseudomonas. (45) The cell according to claim 43, which is a yeast of the genus Saccharomyces. (46) The cell according to claim 44, wherein the bacterium is selected from the group consisting of Bacillus sphaericus, Bacillus subtilis, and Escherichia coli. (47) The cell according to any one of claims 43 to 46, which produces biotin or its vitamin intermediate. (48) A medium containing at least pimelic acid or at least one vitamin intermediate of biotin was added to the bioA gene involved in the bacterial biotin biosynthesis chain, bioD
gene, bioF gene, bioC gene and bio
Fermented by cells permeable to pimelic acid or said biotin vitamin intermediate transformed with a plasmid vector containing at least one DNA sequence encoding an enzyme produced by one of the H genes. A method for producing biotin, which comprises the steps of: (49) Claim 48, wherein the vitamin intermediate of biotin is obtained by fermenting a medium containing at least pimelic acid by the cells that produce the vitamin intermediate. Manufacturing method described. (50) Co-fermentation is carried out using the cell strain that produces a vitamin intermediate of biotin from pimelic acid and the cell strain that ferments this vitamin intermediate to produce biotin. The manufacturing method according to scope item 49. (51) A medium containing at least pimelic acid or at least one vitamin intermediate of biotin is added to one of the following gene groups involved in the bacterial biotin biosynthesis chain: bioF and bioC bioF and bioH bioF, bioC and bioH DN encoding an enzyme produced by
Production of biotin, consisting of fermentation and recovery of the biotin formed by cells permeable to pimelic acid or said vitamin intermediate of biotin, transformed by a plasmid vector containing at least one of the A sequences. Law. (52) Claim 51, wherein the vitamin intermediate of biotin is obtained by fermenting a medium containing at least pimelic acid by the cells that produce the vitamin intermediate. Manufacturing method described. (53) Co-fermentation is carried out using the cell strain that produces a vitamin intermediate of biotin from pimelic acid and the cell strain that ferments this vitamin intermediate to produce biotin. The manufacturing method according to scope item 52.