FR2604436A1 - Cloning of the bioA, bioD and bioF genes of Bacillus sphaericus, vectors and transformed cells and process for preparing biotin - Google Patents

Abstract

The present invention relates especially to a DNA sequence corresponding to one of the following genes of the biosynthetic pathway of biotin in bacteria: bioA gene, bioD gene and bioF gene. Vectors containing these sequences make it possible to transform other microorganisms in order to improve the production of biotin.

Description

 The present invention relates to the preparation of biotin by fermentation using the recombinant DNA technique.

 Biotin is a vitamin necessary for humans, animals, plants and for certain microorganisms.

 It has been isolated from egg yolk and is found in brewer's yeast, cereals, various organs, in free form or combined with proteins.

This vitamin has been proposed in particular as a regulator of skin metabolism, in particular in the treatment of seborrheic dermatitis in humans,
In addition to the various sources which have been mentioned above, biotin is synthesized by certain microorganisms, in particular microorganisms of the genus Bacillus such as Bacillus sphaericus.

 Figure 1 shows schematically the biosynthesis chain of biotin from pimelic acid in this type of microorganism. This biosynthesis chain includes 5 different enzymatic stages in which the genes used are successively called bioC, bioF, bioA, bioD and bioB.

The systematic study of the production of biotin from pimelic acid during industrial fermentations has shown that certain microorganisms, particularly those belonging to the genus
Bacillus sphaericus, hyperproduce vitamins of biotin as well as biotin (hereinafter called "vitamers" the various intermediates leading to biotin).

 For these bacteria, among the precursors of biotin, DTB (desthiobiotin) represents the predominant component which is produced in much greater quantity than the final molecule: biotin.

There is a strong transcription control repressing the synthesis of biotin which depends on the amount of biotin present in the culture medium; this repression occurs in E. coli as well as in
Bacillus sphaericus.

However, no feedback inhibition regulates the biosynthesis of biotin in E. coli and such control has never been described in
Bacillus sphaericus.

 The complete explanation of the production of a very large amount of DTB (for a small amount of biotin) from pimelic acid in Bacillus sphaericus still requires a very important molecular biology study on the organization and regulation of the biosynthetic pathway for biotin in this bacteria.

 Preliminary studies of some of the biosynthetic enzymes extracted and semi-purified from strains of B. sphaericus producing DTB do not reveal significant differences with the enzymes of biotin well known in E. coli.

 However, the production of biotin by industrial fermentation of such strains of Bacillus sphaericus (IFO 3525, NCIB 9370) could become competitive with existing chemical processes if the yield of biotin were improved. In order to achieve this goal, it would be interesting to obtain a constitutive hyperexpression of all the biosynthetic genes of biotin dns C Sç3UCheS.

 We have, of course, described the selection of strains of E. coli that are depressed, either by their resistance to biotin analogs such as alpha-dehydrobiotin, or by selection of mutants hyper secreting biotin. I1 has also been described as "dominant cis" mutations which act plelotropically on the synthesis of all the biotin genes organized into a bipolar operon. Mutations acting in trans have also been mentioned, although, in addition to their power to abolish the transcription control of biotin genes, they often have plelotropic properties which can affect the general physiology of the cell.

 These traditional microbiological selection methods can also be applied to strains of Bacillus sphaericus producing DTB, if it is hypothesized that the molecular control of biotin biosynthesis and the general organization of biotin genes are the same as in E. coli.

 Beyond all these hypotheses, the use of mutant strains in important industrial fermentations and in continuous culture requires the selection of mutants with a very low reversion rate, which represents a difficult task given the current absence of methods. fine genetic analysis in Bacillus sphaericus.

 Another approach could be to clone all genes in the biotin biosynthesis chain from a DTB-producing strain of Bacillus sphaericus and then modify the 5 'control region in vitro so as to suppress the transcription control.

 In addition, cloning and in vitro manipulation of these genes would allow the study of their general organization and the improvement of their transcription in vivo, by known genetic engineering techniques, implying in particular the use of strong promoters. , known to be functional in Bacillus sphaericus strains.

The reintroduction of all these hyperexpressed genes which would no longer be regulated by biotin (but which would be regulated inducibly by using, for example, an induction by an increase in temperature or by an inexpensive substrate) in the original strain of
Bacillus sphaericus could then be implemented using known techniques of transformation, transduction or conjugation-mobilization.

It is also possible to envisage the introduction of these genes into various microorganisms which are known to be acceptable in the food industry and which are permeable to pimelic acid, for example Bacillus subtilis, Saccharomyces cerevisiae or strains of
Pseudomonas.

 Finally, the stabilization of this genetic information in microorganisms could be carried out by a directed integration in the chromosomes or by a self-selection of plasmids as described, for example, in French patent No. 84 12598.

 The cloning and characterization of the bioB gene of Bacillus sphaericus have already been described in patents JP-A-166 992/85 of July 29, 1985, 3P-A-66 532/86 of March 25, 1986 and JP-A-95 724 / 86 of April 24, 1986.

 More particularly, the present invention relates to the DNA sequences coding for the enzyme produced from one of the following genes of the biotin biosynthesis chain in bacteria - bioA gene - bioD gene, and - bioF gene.

 These DNA sequences according to the invention are linked in the form of a "cluster" with the bioB gene which had been previously identified and the whole therefore covers the major part of the biotin biosynthesis chain.

 Although the DNA sequences mentioned above may be of diverse origin, it is preferable to use the sequences originating from a strain of Bacillus, in particular from a strain of Bacillus sphaericus.

 As indicated above, it is particularly interesting that these DNA sequences are devoid of the natural sequences ensuring the control of the transcription of the enzymes of the biotin biosynthesis pathway in the original bacterium in order to abolish the regulation. natural due to biotin and to place these DNA sequences under the control of selected elements, which will ensure their efficient transcription in the host strain.

 The invention relates, in particular, to; Jt or part of the sequences which are represented in FIGS. 4 to 8 and which code for one of the genes mentioned above.

 The DNA sequences according to the present invention can be used in different ways.

 First of all, these sequences can be introduced into a plasmid vector which may or may not have an origin of efficient replication in the host cell.

 When the plasmid vector is non-replicable, it preferably comprises DNA sequences allowing its chromosomal integration in the host strain; to do this, these sequences must be homologous to sequences already present in the genome of the strain to be transformed. These homologous sequences may be natural, that is to say be found in the chromosome, or else have been introduced therein by an integration plasmid, this latter type of plasmid generally comprising a gene for resistance to an antibiotic and a marker gene dependent on a promoter of the strain to be transformed.

 In the context of the present invention, the integration plasmid vector is more particularly the plasmid pTG475 which will be described below and which comprises in particular an inducible promoter.

 When the vector has an origin of autonomous replication, the DNA sequences coding for the enzymes mentioned above will preferably be flanked by control elements ensuring their expression in the host strain; it will be in particular, at the 5 ′ end, a strong promoter, effective in said strain and possibly other elements such as a termination sequence when the host strain is a yeast or any other microorganism such sequence is necessary.

The present invention also relates to the cells transformed by the vector plasmids according to the invention. Among these cells, mention should be made more particularly of bacteria, in particular of the genera Bacillus,
Escherichia or Pseudomonas, but also yeasts, in particular of the genus Saccharomyces.

 Finally, the present invention relates to a process for the preparation of biotin in which a growth medium containing at least pimelic acid, or one of the vitamins of biotin, is fermented by cells as they have been described above, which are permeable either to pimelic acid or to said biotin vitamers and in that the biotin produced is recovered.

Other characteristics and advantages of the present invention will appear on reading the examples below described with reference to the figures in which FIG. 1 represents the biosynthesis chain of biotin, FIG. 2 schematizes the plasmid pTG1400,. FIG. 3 schematizes the plasmid pTG475,. FIG. 4 represents the non-coding sequence upstream of the sequence
LORF nO 1,. FIG. 5 represents the LORF nb I sequence corresponding to the bioD gene, FIG. 6 represents the LORF nO 2 sequence corresponding to the bioA gene, FIG. 7 represents the LORF nO 3 sequence corresponding to the Y gene, FIG. 8 represents the LORF sequence n 4 corresponding to the bioB gene,. FIG. 9 schematizes the complementation study of pTGl400,. Figure 10 shows schematically the complementation study between different
plasmids,. FIG. 11 diagrams the structure of the plasmid pTG1418, FIG. 12 diagrams the complementation test for pTG1418.

EXAMPLE 1
Cloning by complementation of the bioA and bioD genes of Bacillus sphaericus IFO 3525 and demonstration of their link with bioB
a) In E. coli
Bacillus sphaericus IFO 3525 is cultured in 200 ml of PAB culture medium (DIFCO Bacto antibiotic, medium 3, 17.5 g / l) at 37 ° C. for 17 hours. The bacteria are recovered by centrifugation and the total DNA is then extracted from the cells by the Saito method (Saito et al. BBA 1963, 72, 619-629). 450 g of pure DNA are obtained.

 20 μg of the total DNA are placed in total restriction with HindIII (3 U / μg of DNA). pBR322 is treated with alkaline phosphatase after being fully digested with HindIII.

The hybrid recombinant plasmids are obtained by mixing the genomic DNA digested with Hindlil (2 lig) and pBR322 treated as above (I pg) with 2 units of T4 ligase (Boehringer Mannheim)
in 50 l of the reaction buffer containing 30 mM NaCI, Tris HCI pH 7.5
30 mM, MgCl2 10 mM, EDTA pH 8 0.2 mM, DTT 2 mM, ATP pH 7 0.5 mM and
BSA 0.1 mg / ml. The incubation is carried out at 140C for 16 hours. Of
aliquots of the ligation mixture are then added in an experiment of
transformation (Cohen et al. (1972) PNAS 69, 2110-2114) using the
E. coli C600 r K m K + strain and selecting the strains for their
resistance to ampicillin (100 pg / ml> on LB medium.

4 different "pools" of plasmid DNA are then extracted,
each corresponding to an average of 104 individual clones on the
transformation boxes.

Different bio mutants of E. coli are then transformed with these
DNA pools and transformants are selected either in the presence
ampicillin (100 pg / ml) on LB medium either for resistance to this
antibiotic and for prototrophy for biotin at the same time (middle
LB + ampicillin 100 Pg / ml + avidin 0.2 U / ml).

The results observed are collated in Table 1
Table 1
Genotype of the Amp Selection strain. Selection on medium Amp
of E. coli Transformants / g DNA + avidin 0.2 U / ml
DNA transformants / pa
C268 * #bioA, his> 103 3 (pool n 4)
(for each pool) 1 (pool n 1)
C173 * ssbioD, his> 103 2 (pool n 4)
(for each pool)
*: Cleary and Campbell (1972) J. Bacteriol. 112, 830.

The plasmids are isolated from the clones selected on
ampicillin + avidin and analyzed using restriction enzymes.

Three plasmids (2 from strain C268 and 1 from strain C173)
contain a 4.3 kb Hindlil insert with a BglII site and 2 Spahi sites and without a BamHI, Sall, Pstl, EcoRV, Pull, Downstream site.

With one of these plasmids, called pTGl400, and the restriction map of which is shown in FIG. 2, it is possible to retransform by selecting for resistance to ampicillin and prototrophy for biotin, as well the strains C268 (dbioA), C173 ZbioD), R877 (bioDI9) (Cleary and Campbell, 1972) or C162 (bioB) with an average frequency corresponding to that obtained for selection with ampicillin alone (more than 103 per p8 of DNA) . No complement of the auxotrophy for the biotin of the strains R878 (bioC23) or R901 (bbioA-D) (Cleary and
Campbell, 1972) cannot be obtained using pTGl400.

b) In Bacillus subtilis
The complementation of the biological mutants of Bacillus subtilis could prove to be difficult because it is known that deletions can occur with very high frequency in the foreign inserts cloned in the usual replicative plasmids of Bacillus subtilis. This is why a new strategy has been developed based on complementation tests using a non-replicating plasmid. The integration of non-replicative plasmids into the genomic DNA of Bacillus subtilis occurs with a fairly high frequency (approximately 104 transformants / lJg of DNA), using naturally competent cells of Bacillus subtilis, if homologous regions exist between the Genomic DNA and the plasmid.

The first step consists in integrating the plasmid pTG475, the structure of which is indicated in FIG. 3 in various bio mutants of
Bacillus subtilis.

 The plasmid pTG475 contains the XylE gene which codes for the enzyme C2 3 oxygenase (Zukowski et al. (1983) PNAS USA, 80, 1101-1105), which can be used as a chromogenic marker (yellow) and which is expressed under the control of the inducible promoter of the levan sucrase gene from Bacillus subtilis. This plasmid also contains a CAT gene conferring resistance to chloramphenicol; the CAT and XylE genes are inserted into pBR322.

This plasmid is integrated into the chromosome of the following strains of Bacillus subtilis:. bioA strain: JKB 3173 (bioA 173, aro G932; CH Pai (1975)
J. Bacteriol. 121, 1-8), bioB strain: BGSC1A92 (bioB 141, aro G932 Sac A 321, Arg A2
CH Pai (197S) J. Bacteriol. 121, 1-8, .soul bioll2: JKB 3112 (bio 112; CH Pai (1975) J. Bacteriol. 121, 1-8), by the technique of transformation of competent cells of RJ

Boyland (1972) J. Bacteriol. 110, 281-290, the selection being made on
TBAB (DIFCO Blood tryptose agar base)) plus chloramphenicol 3 pg / ml.

Different checks are carried out on the transformed clones
they turn yellow when induced with sucrose, and further a Southern hybridization control for the bioA, bioB and bioll2 strains shows that the integration of pTG475 by simple recombination in the promoter of the levan sucrase gene has taken place. . These transformed strains of Bacillus subtilis are called bioA TG1, bioB TG2 and biol 12 TG3. The plasmid pTG475 having brought sequences of pBR322 into the genome of Bacillus subtilis, it becomes possible to integrate, by homologous recombination, any foreign plasmid comprising these same sequences.

 In a second step, the plasmid pTG1400, previously cloned by complementation in E. coli, was used to transform the strains of Bacillus subtilis bioA TG1, bioB TC2 and bioll2 TG3. The selection is carried out on LB medium + avidin 0.2 U / ml + chloramphenicol 10 pg / ml.

 It is observed that it is possible to select, at a very high frequency, prototrophic transformants for biotin from bioA TG1 and bioB TG2 strains but not with the bioll2 TC3 strain.

pTG1400 therefore does not complement the bioll2 mutation.

c) Final characterization of the Hindlil insert of pTG1400
Southern hybridization experiments were carried out in order to detect the same HindIII fragment in the genomic DNA of
Bacillus sphaericus IFO 3525, corresponding to the insert of pTG1400.

Under drastic hybridization conditions (50% formamide, 0.6% Denhardt solution, 0.1% SDS, 3xSSC at 420C) and using a plasmid pTG1400 labeled with 32p by incorporation of radioactive nucleotides by in vitro polymerization ("nick translation") (2.5.1 07 cpm / ug of DNA), a single Hindlil band of 4.3 kb could be visualized in the genomic DNA of Bacillus sphaericus IFO 3525 after 6 hours of autoradiography at -800C. It has been verified that under these hybridization conditions
pBR322 did not cross-react with genomic DNA from
Bacillus sphaericus IFO 3525. Under the same conditions, no positive reaction, with pTG1400 as probe, could be detected in the genomic DNA of Bacillus subtilis BGSC1A289 treated with Hindili or in the genomic DNA of E. coli C600 treated with Hindlil.

 The total DNA sequence of the 4.3 kb HindIII fragment was analyzed using the "Shotgun" method, that is to say the systematic cloning of the fragments obtained by sonication, the "cyclone deletions" or the elongations with oligonucleotide-primers.

 For the "Shotgun" method, the plasmid pTG1400 was cut by treatment with ultrasound. After treatment of the DNA segments with the DNA polymerase of phage T4, the blunt-ended fragments are allowed to migrate on a low-melting agarose gel and fragments of approximately 300 bp are isolated.

The cloning of these fragments was carried out in M13 vectors digested with 5 May and treated with phosphatase,
The white areas which do not give cross hybridization with pBR322 are sorted and 100 clones are sequenced. The results were analyzed by computer.

 A recent method for obtaining a series of overlapping clones (the cyclone system) was used for DNA sequencing (RMK Dale, BA Mc Clure, JP Houchins (1985) Plasmid 13, 31-40) . This method was continued in parallel with the "Shotgun" method, in order to confirm the results. In addition, this method produces defined deleted plasmids containing groups of bio genes or isolated bio genes.

 The complete sequence of the HindIII fragment of pTC1400 is shown in Figures 4, 5, 6, 7 and 8.

 Computer analysis of this sequence reveals that the fragment has the capacity to code for four long open reading frames (LORF). The possible translation initiation sites and the Shine and Dalgarno regions are underlined. A palindromic region is underlined at the 3 'end of the sequence which could represent a transcription termination site.

 The detailed complementation analysis shows that the first LORF region (FIG. 9) (with 3 possible translation initiation sites) corresponds to the bioD gene.

 Experiments known as "maxicells" were carried out with the strain of E. coli CSR 603 (recAl, phr-l, uvrA6, thr-l, leu-6, thi-l, argE3, lakes1, galK2, aral4 , xyll5, mtll, proA2, str-31, tsx-33, supE44, F-, lambda); they show that this region codes for a polypeptide with an apparent molecular weight of approximately 25 kd.

 The second LORF region (Figure 6) (with 4 possible translation initiation sites) corresponds to the bioA gene. An experiment in "maxicells" of E. coli CSR603 reveals a polypeptide with an apparent molecular weight of approximately 40 kd which corresponds to the product of the bioA gene.

 It was not possible to determine the function of the third LORF sequence, called the Y gene (Figure 7). A very high hydrophobicity and the presence of a probable signal sequence in the coding region suggest that this LORF, if it is transcribed and translated, codes for a protein having an interaction with the membrane. The fact that this Y gene is in a "cluster" with the other bio genes (A, D and B) suggests that it codes for another function involved in the metabolism of biotin.

 The fourth LORF region (FIG. 8) (with 3 possible translation initiation sites) has already been identified, it is a region coding for the bioB gene. It is demonstrated here that this gene is linked in a "cluster" with the bioA and bioD genes.

Complementation analysis with plasmids containing subcloned regions of the 4.3 kb Hindlil insert of pTG1400 clearly shows, as shown in Figure 9, that the first region
LORF corresponds to the product of gene D and that the second LORF region corresponds to the product of gene A.

EXAMPLE 2
Cloning by complementation of the bioF gene of Bacillus sphaericus IFO 3525
The Hindlil genomic DNA library of Bacillus sphaericus IFO 3525 described previously was used to transform an mutant of E.

coli, bioF 12, following the methodology described above.

The results obtained are collated in Table 2
Table 2
Genotype of the Amp Selection strain. Middle selection
of E. coli Amp. + avidin 0.2 U / ml
Transformants / g DNA Transformants / ug DNA
R874 * bioF 12, his> 104 2 (pool n 1)
(for each pool) 2 (pool 3)
4 (pool 4) * Cleary and Campbell (1972) J. Bacteriol. 112, 830
The plasmids are isolated from the clones selected on medium containing ampicillin and avidin ':: analyzed using restriction enzymes.

Two of these plasmids contain two Hindili inserts of approximately 4.5 and 0.6 kb with Sphl, Kpnl, HpaI, NdeI, Aval, Clapi, Bgl, XmnI sites,
Pvull, Scal, Stul and without site Bill, Xbal, 5mai, Pstl, Sali, NruI, BamHI, Pvul, EcoRI, HindII, EcoRV, FspI, Apax, Ball, Aatil ..

 With one of these plasmids, pTG1418, it was possible to retransform by selecting for resistance to ampicillin and prototrophy for biotin, the strain R874 of E. coli (bioF12, his) with an average frequency corresponding to that obtained for the selection of clones on ampicillin (more than 104 μg of DNA).

 Subcloning experiments have shown that only the 4.5 kb HindIII fragment codes for the KAPA synthetase function ensuring the complementation of the bioF12 mutation of the E. coli strain R874.

"Southern" experiments were carried out in order to detect the Hindi fragment !! of 4.5 kb in the genomic DNA of Bacillus sphaericus IFO 3525 in comparison with the insert of 4.8 kb of pic1418. Using very drastic hybridization conditions (50% formamide, 3xSSC, 0.1% SDS, 0.6% Denhardt's solution, 429C) and using
M13TG1425 (phage M13 containing the Hindi !! insert of 4.5 kb labeled with 32p by incorporation of radioactive nucleotides by in vitro polymerization ("nick translation)" with 2.106 cpm / pg of DNA), a Hindi band !! about 4.5 kb can be seen in the genomic DNA of Bacillus sphaericus
IFO 3525 after 7 hours of autoradiography at -80 "C.

 Under the same conditions, no positive reaction can be detected, nor on the genomic DNA of Bacillus subtilis BGSClA289 treated with Hindi !! nor on the genomic DNA of E. coli C600 treated by Hindi !!.

 No cross-reaction can be detected between the insert of pTG1400 and the inserts of pTG1418, neither with an 8.3 kb Spahi fragment of pTG1406 overlapping the 3 'end of pTG1400, nor with an 8.2 kb MboI fragment of pSBOl overlapping the 5 'end of pTG1400 (see Figure 10).

 The restriction map of the inserts of pTG1418 has been analyzed and is indicated in FIG.

 Complementation studies and "Southern" analysis demonstrate that the bioF gene from Bacillus sphaericus IFO 3525 is not linked to the bioA, bioD and bioB genes of the same microorganism (Figure 12).

Deposit of strains representative of the invention
The E. coli C600 pTG1400 and R874 pTG1418 strains were deposited at the National Collection of Cultures of Microorganisms of the Institut Pasteur, 28 rue du Docteur-Roux - 75724 Paris Cédex 15, September 26, 1986 under numbers 1-608 and 1-609.

REFERENCES
PP Cleary and A. Campbell (1972) J. Bacteriol. 112, 830-839.

MM Zukowski, DF Gaffney, D. Speck, M. Kauffmann, A. Findeli,
A. Wisecup and JP Lecocq (1983) PNAS USA 80, 1101-1105.

C.H. Pai (1975) 3. Bacteriol. 121, 1-8.

 R.J. Boyland, N.H. Mendelson, D. Brooks and F.E. Young (1972) J. Bacteriol.

110, 281-290.

R.M.K. Dale, B.A. Mc Clure, J.P. Houchins (1986) Plasmid 13, 31-40.

S.N. Cohen, A.C.Y. Cheng, L. Hsu (1972) PNAS USA 69, 2110-2114.

H. Saito and K.I. Miura (1963) BBA 72, 619-629.

Claims (24)

 1) DNA sequences coding for the enzyme produced from one of the following genes in the biotin biosynthesis chain in bacteria - bioA gene, - bioD gene and - bioF gene.  2) DNA sequence according to claim 1, characterized in that it codes for the enzymes produced from the D gene, A gene and B gene.  3) DNA sequence according to one of claims 1 and 2, characterized in that it codes for the enzymes produced from the D gene, gene A, B gene and F gene.  4) - DNA sequence according to one of claims I to 3, characterized in that it comes from a strain of Bacillus.  5) DNA sequence according to claim 4, characterized in that it is the bioA gene, bioD gene or bioF gene which comes from a strain of Bacillus sphaericus.  6) DNA sequences according to one of claims 1 to 5, characterized in that they are devoid of natural sequences ensuring the control of the transcription of the enzymes of the biosynthetic pathway of biotin in the original bacterium.  7) DNA sequence according to one of claims I to 5, characterized in that it corresponds to all or part of the sequence described in Figures 4 to 8.  8) Plasmid vector comprising at least one sequence according to one of claims 1 to 7.  9) Vector according to claim 8, characterized in that it is a non-replicable vector which comprises the DNA sequences allowing its integration into a chromosome in the host strain.  10) Vector according to claim 9, characterized in that it comprises at least one sequence homologous to a sequence present in the genome of the strain to be transformed and ensuring integration.  11) Vector according to claim 10, characterized in that the homologous sequence is a natural genomic sequence.  12) Vector according to claim 10, characterized in that the homologous sequence is a sequence which has been integrated into the genome thanks to a plasmid integration vector.  13) Vector according to claim 12, characterized in that the specific plasmid vector comprises at least one gene for resistance to an antibiotic and a marker gene under the dependence of a promoter of the strain to be transformed.  14) Vector according to claim 13, characterized in that the promoter is an inducible promoter.  15) Vector according to claim 14, characterized in that the integration plasmid vector is the plasmid pTG475.  16) Vector according to claim 8, characterized in that it comprises an origin of autonomous replication.  17) Vector according to claim 16, characterized in that 'it is an origin of autonomous replication in a Bacillus strain or an Escherichia strain.  18) Vector according to one of claims 16 and 17, characterized in that the DNA sequences coding for the enzymes of the biotin biosynthesis chain are flanked by elements ensuring their expi ession in the host strain.  19) Vector according to claim 18, characterized in that among the elements ensuring their expression is an effective promoter and terminator in the host strain.  20) Cells transformed by a vector according to one of claims 8 to 19.  21) Cell according to claim 20, characterized in that it is a bacterium chosen from the genera Bacillus, Escherichia or Pseudomonas.  22) Cell according to claim 20, characterized in that it is a yeast of the genus Saccharomyces.  23) Cell according to claim 21, characterized in that the bacteria is chosen from Bacillus sphaericus, Bacillus subtilis and Escherichia coli.  24) Process for the preparation of biotin, characterized in that a culture medium containing at least pimelic acid or one of the biotin vitamers is fermented by cells according to one of claims 20 to 23 , permeable to pimelic acid or the so-called vitamin of biotin and in that the biotin recovered is recovered.