FR2615514A2 - Cloning of the bioC and bioH genes of Bacillus sphaericus, vectors and transformant cells and process for preparing biotin - Google Patents

Abstract

The present addition relates to a DNA sequence according to one of Claims 1 and 4 to 6 of the parent patent, characterised in that it encodes the enzymes which are products of the genes: . bioF and bioC . bioF and bioH . bioF, bioC and bioH.

Description

 The present addition relates to the cloning and characterization of genes corresponding to the first steps of biotin biosynthesis in bacteria.

 The main patent describes the cloning and expression of DNA sequences encoding the bioA, bioD and bioF genes of biotin biosynthesis.

 As described in the main patent, the use of expression vectors carrying the above genes makes it possible to transform bacteria into biotin from different vitamins of this product.

 However, the preparation of biotin can be considered in a truly economical way on the industrial level only if the starting vitamin is pimelate, that is why it was necessary to be able also to ensure the expression of the genes corresponding to the first stages of the process. biosynthesis of biotin in bacteria, namely. the bioC gene, and. the bioH gene.

 This is why the present addition relates, first of all, to DNA sequences conforming to the main patent characterized in that they encode the produced gene enzymes: bioF and bioC. bioF and bioH. bioF, bioC and bioH.

 More particularly, the present addition relates to a DNA sequence, characterized in that it corresponds to all or part of the sequence described in FIGS. 2, 3 and 4.

 In general, the present addition relates to DNA sequences characterized in that they code for the produced enzymes of the H gene or C gene.

 Preferably, the DNA sequences coding for the produced enzymes of the H gene or the C gene will also carry the sequences coding for one of the genes described above, namely the bioA gene the the bioD gene the bioF gene.

 As has been previously described in the main patent, preferably the DNA sequences in question may be carried by a plasmid vector capable of ensuring the transformation of a bacterium and comprising all the elements ensuring the expression of genes correspondents.

 The ceplasmide may, as indicated, be of autonomous type and self-replicable or, on the contrary, be non-replicable but intended to ensure its integration into the chromosome of the host strain.

 To do this, the different technologies to be implemented have been described in the main patent.

 Thus, in the case of a non-replicable integration vector, the latter must comprise at least one homologous sequence of a sequence present in the genome of the souchi to be transformed, which will enable chromosomal integration to be ensured. . It can obviously be either a homologous sequence corresponding to a natural genomic sequence or a homologous sequence introduced by another plasmid integration vecteu.

 Similarly, these different plasmid vectors may include elements ensuring a selection such as an antibiotic resistance gene and / or a marker gene, under the control of a promoter of the strain to be transformed.

Among the cells that can serve as a host strain, mention should be made more particularly of bacteria, in particular of the genera Escherichia, Bacillus and Pseudomonas, as well as yeasts, in particular yeast of the genus
Saccharomyces.

 Among the particularly interesting host cells are Bacillus sphaericus, Bacillus subtilis and Escherichia coli.

Finally, the strains of particular interest for being transformed by the DNA sequences according to the present addition, are the strains which have already been transformed by vectors ensuring the expression of the other genes of this biosynthetic pathway, that is to say say the F genes,
A, D and B as described in the main patent.

 Under these conditions, the introduction of the bioC and bioH genes in the bacterium allows this bacterium to synthesize biotin from the first vitamin in the chain, namely the pimelate, which is of significant economic interest on the industrial level.

The following examples are more particularly intended to highlight other advantages and characteristics of the present addition. The following figures complete the examples. FIG. 1 represents the restriction map of the insert.
B. sphaericus used in the following plasmids. Figure 1b schematizes plasmids pTG1418 and pTG1420. Figure 1c schematizes the plasmids pTG1422 and pTG1435 Figure 1d schematizes the plasmids pTG1436 and pTG1437. Figure 2 shows the LORF X sequence. FIG. 3 represents the LORF sequence. FIG. 4 represents the LORF sequence F. Figure 5 schematizes the plasmid pTG1440. FIG. 6a represents the restriction card of the insert
plasmid pTG1418. Figures 6b and 6c show the different plasmids
pTG1418 derivatives used in complementation tests
tion.

Example 1 Complementation of the B. subtilis bioll2 mutant
TG3 (derived from JKB 3112, bioC / F-112 aroG932) by
integrative transformation with pTGl4l8 and dif
derived from it.

 The bioll2 mutant of B. subtilis has been identified by nutritional tests as affected in the bioF or bioC function (C.H. Pai, 1975, J. Bacteriol 121, 1-8). In c mutant, plasmid pTG475 was integrated at the R-bag B bag locus, following the methodology described in the main breve; the new strain thus obtained was denoted bioll2 TG3.

Transformation of the bioll2 TG3 strain by different plasmids (pTG1418, 1420, 1422, 1435, 1436 and 1437, shown in Figure 1a-1d) was performed, followed by selection on LB + chloramphenicol medium 10 μg / ml. ml + avidine 500 U / 1. The complementation results are summarized in Table 1. As the crossover test with the mutant
R874 (bioF, his) of E. coli gives the same result, it is likely that the bioll2 mutant of B. subtilis is affected in the bioF gene. By analyzing the complementations obtained according to the plasmids used, it is possible to locate the bioF gene on the pTG1418 insert at a fragment delimited by the XmnI and NcoI restriction sites (see FIG. 6).

Table 1 Integrative transformation of the strain of
B. subtilis bioll2 TG3.

<Tb>

Plasmid <SEP> Insert <SEP> complementation <SEP> on <SEP> LB <SEP> +
<tb><SEP> 500 <SEP> U / 1 <SEP> avidin <SEP> + <SEP> 10 <SEP> y / ml
<tb><SEP> 8 <SEP> chloramphenicol
<tb><SEP> Insert <SEP> HindIII <SEP> of <SEP> ++ <SEP> I
<tb><SEP> 5.1 <SEP> kb
<tb> pTG1422 <SEP> Insert <SEP> ClaI <SEP> of <SEP> 3.1 <SEP> ++
<tb><SEP> kb <SEP> containing <SEP> bioF <SEP> and
<tb><SEP> a <SEP> part <SEP> of <SEP> LORF <SEP>t;;<September>
<tb> pTG1420 <SEP> Insert <SEP> ClaI-HindIII <SEP>
<tb><SEP> of <SEP> 2 <SEP> kb <SEP> containing <SEP> X
<tb><SEP> and <SEP> a <SEP> part <SEP> of
<tb><SEP> LORF <SEP> 11 <SEP>
<tb> pTG1436 <SEP> Insert <SEP> XmnI-NcoI <SEP> of <SEP> ++
<tb><SEP> 1.3 <SEP> kb <SEP> containing <SEP> the
<tb><SEP> gene <SEP> bioF
<tb> pTG1437 <SEP> Insert <SEP> NcoI-XmnI <SEP> of <SEP> ++
<tb><SEP> 1.3 <SEP> kb <SEP> containing <SEP> the
<tb><SEP> gene <SEP> bioF
<tb> pTG1435 <SEP> Insert <SEP> HpaI-PvuII <SEP> of
<tb><SEP> 0.6 <SEP> kb <SEP> containing <SEP> the
<tb><SEP> LORF <SEP> X
<Tb>
Example 2 Nucleotide Sequence of the HindIII Insert of
4.53 kb of plasmid pTG1418.

 The HindIII insert was cloned into M13TG131 at the corresponding site of the polylinker. The so-called "cyclone" method was applied to this plasmid M13TG1425 and the results analyzed by computer. The few differences in reading between the two complementary strands were cleared by sequencing using specific oligonucleotides.

 The sequence is detailed in Figures 2 & 4. It appears that this insert has three open reading frames, encoding respectively proteins (subunits) of a molecular weight of 18462, 28048 and 42940 daltons. The last gene, relative to the sense of reading, is located in the region identified as bioF, the molecular weight of this protein of B. sphaericus being of the same order of magnitude as that of KAPA synthetase of E. coli (M.k.

Eisenberg, 1973, Adv. Enzymol 38, 317-372).

 The juxtaposition of the three open reading frames could, as in the case of the insert of the plasmid pTG1400, be typical of an operon structure. It should be noted that it is possible to identify, upstream of the first gene, a sequence of fifteen base pairs (underlined in FIG. 2 also present upstream of the first gene (bioD) of the insert of plasmid pTG1400. significant could indicate that both groups of biotin B sphaericus genes are subject to at least one common regulation.

This could correspond to control by biotin (o a derivative of this one), as already described for the
KAPA synthetase (bioF) from B. sphaericus (Y. Yzumi, K. Sato,
Y. Tani and K. Ogata, 1973, Agric. Biol. Chem. 37, 1335).

On the 3 'side of the last gene of the sequenced insert, a sequence having the characteristics of a transcription terminator can be identified (underlined in the figure).
Example 3 Complementation of mutants E. coli R878 (bioC,
his) and C261 (bioFCD, his) respectively, at
using plasmids pTG1418 (1433) and pTG1440.

 The traditional methodology of complementing E. coli bio mutants was applied using plasmid pTG1418 and various derivatives thereof.

When competent cells of the E. coli mutant
R878 (bioC, his) are transformed with the plasmids pTG1418 and pTG1433 (see Table 2), and then spread on medium
LB + ampicillin 100) Ig / ml + avidin 200 U / 1, growth is detected after 36 h incubation at 37 μl. The frequency of appearance of the clones transformed on this medium is of the same order of magnitude as that measured on the medium LB + ampicillin 100 Rg / ml.

Table 2: Transformation of E. coli mutant R878 bioC. Name-
number of transformants per microgram of DNA.

<Tb>

Plasmid <SEP> Selection <SEP> on <SEP> Medium <SEP> Selection <SEP> on <SEP> Medium
<tb><SEP> LB <SEP> + <SEP> ampicillin <SEP> LB <SEP> + <SEP> ampicillin <SEP> + <SEP> avidin
<tb><SEP> (100 <SEP> μg / ml) <SEP> (100 <SEP> Rg / ml) <SEP> (200 <SEP> U / 1)
<tb> pTG1418 <SEP> 103 <SEP> 103 <SEP> small
<tb> pTG1433 <SEP> 103 <SEP> 103 <SEP> small
<tb> pBR322 <SEP> 103 <SEP> 0 <SEP>
<Tb>
After 36 h incubation at 37 ° C
Growths of transformed clones, normal on
LB + ampicillin are slowed down in the absence of biotin (LB medium + ampicillin + avidin, LB + avidin, minimum plus casamino acids, devoid of biotin) .This complementation of biotin auxotrophy mutant R878 bioC is all-to- significantly, given the total absence of residual growth of the same mutant, when it is transformed with various plasmids derived from pBR322, on medium lacking biotin.

Both inserts of plasmids pTG1400 and pTG1418 were cloned into pBR322 to give plasmid pTG1440 (see Figure 5). This plasmid pTG1440, when introduced into the E. coli mutant C261 (bioFCD, his), allows the selection of clones on LB medium + ampicillin 100; sg / ml + avidin 200 U / 1. The transformation frequency obtained is directly comparable to that measured on LB + ampicillin. Again, the growth of these recombinant clones, normal on LB + ampicillin medium, is slowed down in the absence of biotin. From these two results (complementation of the biotin auxotrophy of the mutant bioC and bioAFCD), it is clear that the insert of the plasmid pTG1418 also contains the B. sphaericus bioC gene. The different subclonings derived from the pTG1418 (see FIG. 6) do not make it possible to obtain the complementation of the bioC mutation of
E. coli. Only the inserts possessing the three genes confer an effective complementation of the bioC mutation of
E. coli.

Example 4 Complementation of the mutant E. coli bioH (PA505
MAt108, argH, metA, bioH, malA, strr).

 This mutant was originally described as bioB (D.

Hatfield, M. Hofnung and M. Schwartz, 1969, J. Bacteriol. 98, 559-567). It was then characterized as nonexistent and able to grow on mineral medium in the presence of KAPA, DAPA, DTB or biotin. Eisenberg (1985,
Annals New York Academy of Sciences 447, 335-349) then proposed that this gene encodes a subunit of pimeloyl
CoA synthetase (bioH). It should be noted that the mutants of
Overproducing E. coli of biotin (selected either by a level of vitamin excretion allowing the growth of an E. coli bioB auxotroph or by resistance to adéhydrobiotine) have all been genetically identified as assigned to the bioR locus. This locus encodes a multifunctional protein (repressor of messenger RNA synthesis of the bioABFCD operon and biotin-binding holoenzyme synthetase on a lysine residue of different carboxylase-functional apoenzymes).

 The fact that all the biotin-producing E. coli mutants identified today are localized in the gene encoding the trans-active repressor and never in the operator of the bioABFCD operon, suggests that another gene biosynthesis of biotin is subject to this regulation. In terms of literature, the best candidate is the bIoH gene.

The pTG1418 insert containing the bioF and bioC genes from
B. sphaericus, it was investigated whether the third gene corresponded to bioH.

 Complementation of the E. coli bioH mutant was effectively achieved using plasmid pTG1433. Once again, the growth obtained on this medium is slowed down, but quite significant compared with the controls (see Table 3).

Table 3: Transformation of the mutant E. coli bioH PA505
t4AtlO8. Number of transformants per microgram
DNA.

<Tb>

Plasmid <SEP> Selection <SEP> on <SEP> Medium <SEP> Selection <SEP> on <SEP> Medium
<tb><SEP> LB <SEP> + <SEP> ampicillin <SEP> LB <SEP> + <SEP> ampicillin <SEP> + <SEP> avidin
<tb><SEP> (100 <SEP> Fg / ml) <SEP> (100 <SEP> Fg / ml) <SEP> (200 <SEP> U / 1)
<tb> pBR322 <SEP> 104 <SEP> 0
<tb> pTG1433 <SEP> 103 <SEP> 3 <SEP><SEP> 103 <SEP> 3 <SEP> small
<tb><SEP>#
<Tb>
after 36 hours of incubation
at 37'C
As for the complementation of the mutant block, a necessary and sufficient condition for the complementation of the mutant bioH is the simultaneous presence of the three genes of the pTG1418 insert on the plasmids introduced into the strain (see FIG. 6).

In the case of E. coli bioF mutants and
B. subtilis, which can be complemented by a single B gene.

sphaericus, the growth in the absence of biotin mutants bioC and bioH E. coli can therefore be obtained only during the introduction of recombinant plasmids carrying the three genes of the HindIII insert of pTG1418. This could reflect, among other things, differences in enzymatic properties between B. sphaericus pimeloyl-CoA synthetase and the corresponding E. coli enzyme (difference in affinity for substrates, multienzyme educt, etc.). .) or reduced synthesis of B. sphaericus pimeloyl-CoA synthetase in
E. coli, limiting the metabolic flux of the pimelate to the
KAPA.

Claims (21)

 1) DNA sequence according to one of claims 1 and 4 & 6 of the main patent, characterized in that it encodes for the produced gene enzymes. bioF and bioC bioF and bioH. bioF, bioC and bioH.  2) DNA sequence characterized in that it corresponds to all or part of the sequence described in Figures 2, 3 and 4.  3) DNA sequence according to claim 2, characterized in that it codes for the produced enzymes of bioH gene or bioC gene.  4) DNA sequence according to one of claims 1 to 3, characterized in that it further codes for at least the enzyme produced by one of the following genes. bioA gene. bioD gene. bioF gene.  5) Plasmid vector comprising at least one sequence according to one of claims 1 to  6) Vector according to claim 5, characterized in that it is a non-replicable vector which comprises the DNA sequences for its integration into a chromosome in the host strain.  7) vector according to claim 6, characterized in that it comprises at least one homologous sequence of a sequence present in the genome of the strain to be transformed and ensuring the integration.  8) Vector according to claim 7, characterized in that the homologous sequence is a natural genomic sequence.  9) Vector according to claim 7, characterized in that the homologous sequence is a sequence which has been integrated into the genome by means of an integration plasmid vector.  10) Vector according to claim 9, characterized in that the specific plasmid vector comprises at least one antibiotic resistance gene and a marker gene under the control of a promoter of the strain to be transformed.  11) Vector according to claim 10, characterized in that the promoter is an inducible promoter.  12) Vector according to claim 11, characterized in that the plasmid integration vector is the plasmid pTG475.  13) Vector according to claim 5, characterized in that it comprises an autonomous origin of replication.  14) Vector according to claim 13, characterized in that it is an autonomous origin of replication in a strain of Bacillus or a strain of Escherichia.  15) Vector according to one of claims 13 and 14, characterized in that the DNA sequences encoding the enzymes of the biotin biosynthetic chain are flanked by elements ensuring their expression in the host strain  16) A vector according to claim 15, characterized in that among the elements ensuring their expression is an effective promoter and terminator in the host strain.  17) cells transformed with a vector according to one of claims 5 & 16.  18) Cell according to claim 17, characterized in that it is a bacterium selected from the genera Bacillus, Escherichia or Pseudomonas.  19) Cell according to claim 17, characterized in that it is a yeast of the genus Saccharomyces.  20) Cell according to claim 18, characterized in that the bacterium is selected from Bacillus sphaericus, Bacillus subtilis and Escherichia coli.  21) Process for the preparation of biotin, characterized in that a culture medium containing at least pimelic acid or one of the vitamers of biotin is fermented by cells according to one of claims 17 to 20, permeable to pimelic acid or said vitamin of biotin and in that recovering the biotin formed.