U.S. patent number RE38,331 [Application Number 09/851,950] was granted by the patent office on 2003-11-25 for cells with altered betaine catabolism and their use in producing metabolites or enzymes.
This patent grant is currently assigned to Rhone-Poulenc Biochimie. Invention is credited to Beatrice Cameron, Joel Crouzet.
United States Patent |
RE38,331 |
Cameron , et al. |
November 25, 2003 |
Cells with altered betaine catabolism and their use in producing
metabolites or enzymes
Abstract
Cells with an alteration at least in the gene involved in
betaine catabolism, their preparation and their use, in particular
for producing metabolites and/or enzymes, are disclosed.
Inventors: |
Cameron; Beatrice (Paris,
FR), Crouzet; Joel (Sceaux, FR) |
Assignee: |
Rhone-Poulenc Biochimie
(Antony, FR)
|
Family
ID: |
9436376 |
Appl.
No.: |
09/851,950 |
Filed: |
May 9, 2001 |
PCT
Filed: |
December 07, 1993 |
PCT No.: |
PCT/FR93/01202 |
PCT
Pub. No.: |
WO94/13813 |
PCT
Pub. Date: |
June 23, 1994 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
436393 |
May 18, 1995 |
05691163 |
Nov 25, 1997 |
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Foreign Application Priority Data
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|
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|
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Dec 9, 1992 [FR] |
|
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92 14814 |
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Current U.S.
Class: |
435/41; 435/183;
435/189; 435/190; 435/252.3; 435/253.3; 435/61; 435/69.1;
435/86 |
Current CPC
Class: |
C12N
15/52 (20130101); C12N 15/68 (20130101); C12N
15/74 (20130101); C12P 19/42 (20130101) |
Current International
Class: |
C12P
19/00 (20060101); C12P 19/42 (20060101); C12N
15/52 (20060101); C12N 15/67 (20060101); C12N
15/68 (20060101); C12N 15/74 (20060101); C12P
001/00 (); C12P 033/02 (); C12P 021/06 (); C12N
009/00 (); C12N 009/02 () |
Field of
Search: |
;435/41,86,253.3,183,61,69.1,440,189,190 ;431/252.3 |
Foreign Patent Documents
Other References
Kawahara, Y. et al., "Effect of glycine betaine an osmoprotective
compound on the growth of Brevibacterium lactofermentum," Appl.
Microbiol. Biotechnol., vol. 33, pp. 574-577 (1990). .
Lago, B.D. et al., "Alternate requirement for vitamin B12 or
methionine in mutants of Pseudomonas denitrificans, a vitamin
B12-producing bacterium," J. Bact., vol. 99, No. 1, pp. 347-349
(Jul. 1969). .
White, R.F. et al., "Betaine-homocysteine transmethylase in
Pseudomonas denitrificans a vitamin B12 overproducer," J. Bact.,
vol. 113, No. 1, pp. 218-223 (1973)..
|
Primary Examiner: Achutamurthy; Ponnathapu
Assistant Examiner: Rao; Manjunath N.
Attorney, Agent or Firm: Finnegan, Henderson, Farabow,
Garrett & Dunner LLP
Claims
We claim:
1. A process for the production of metabolites or enzymes or both
comprising a) genetically modifying a cell by gene disruption,
wherein said disruption causes at least one stable or nonreversible
modification in a gene encoding a protein involved in the
catabolism of glycine-betaine; and b) culturing said cell under
production conditions.
2. The process according to claim 1, wherein the modified cell
degrades betaine less rapidly than an unmodified cell.
3. The process according to claim 2, wherein the modification
affects the coding portion of said gene .[.and/or.]. .Iadd.or
.Iaddend.the regions responsible for the expression .[.and/or.].
.Iadd.or .Iaddend.the transcriptional regulation of said gene.
4. The process according to claim 2, wherein the cell is of a genus
selected from the group consisting of Pseudomonas, Streptomyces,
Actinomycetes, Propionibacterium, Corynebacterium, Bacillus,
Escherichia, Salmonella, Rhizobium, Agrobacterium,
Rhodopseudomonas, Xanthomonas, Clostridium and
Methanobacterium.
5. The process according to claim 2, wherein the metabolite is
selected from the group consisting of (a) amino acids, (b)
vitamins, (c) antibiotics, (d) a derivative of (a), (b) or (c), and
(e) a precursor of (a), (b) or (c).
6. The process according to claim 5, characterized in that the
metabolite is vitamin B12.
7. The process according to claim 1, wherein the cell is of a genus
selected from the group consisting of Pseudomonas, Streptomyces,
Actinomycetes, Propionibacterium, Corynebacterium, Bacillus,
Escherichia, Salmonella, Rhizobium, Agrobacterium,
Rhodopseudomonas, Xanthomonas, Clostridium and
Methanobacterium.
8. The process according to claim 1, wherein the metabolite is
selected from the group consisting of (a) amino acids, (b)
vitamins, (c) antibiotics, (d) a derivative of (a), (b) or (c), and
(e) a precursor of (a), (b) or (c).
9. The process according to claim 8, characterized in that the
metabolite is a cobalamin.
10. The process according to claim 8, wherein the metabolite is
vitamin B12.
11. The process according to claim 1, wherein said gene encodes an
enzyme selected from the group consisting of betaine homocysteine
methyltransferase, dimethylglycine dehydrogenase, and sarcosine
dehydrogenase.
12. The process according to claim 11, wherein said enzyme is
betaine homocysteine methyltransferase.
13. The process according to claim 11, wherein said enzyme is
dimethylglycine dehydrogenase.
14. A process according to claim 1, wherein the step of gene
disruption comprises insertion of a transposon.
15. A process according to claim 1, wherein the step of gene
disruption comprises homologous recombination.
16. A recombinantly modified cell comprising at least one stable or
nonreversible modification in a gene encoding a protein involved in
the catabolism of glycine-betaine, wherein said modification is a
gene disruption.
17. The recombinantly modified cell according to claim 16, wherein
said gene encodes an enzyme selected from the group consisting of
betaine homocysteine methyltransferase, dimethylglycine
dehydrogenase, and sarcosine dehydrogenase.
18. The recombinantly modified cell according to claim 17, wherein
said enzyme is betaine homocysteine methyltransferase.
19. The recombinantly modified cell according to claim 17, wherein
said enzyme is dimethylglycine dehydrogenase.
20. A cell according to claim 16, wherein said gene disruption
comprises an insertion..Iadd.
21. A process for the production of metabolites or enzymes or both,
comprising culturing the recombinantly modified cell according to
claim 16 under production conditions..Iaddend..Iadd.
22. The process according to claim 21, wherein said gene encodes an
enzyme selected from the group consisting of betaine homocysteine
methyltransferase, dimethylglycine dehydrogenase, and sarcosine
dehydrogenase..Iaddend..Iadd.
23. The process according to claim 22, wherein said enzyme is
betaine homocysteine methyltransferase..Iaddend..Iadd.
24. The process according to claim 22, wherein said enzyme is
dimethylglycine dehydrogenase..Iaddend..Iadd.
25. The process according to claim 21, wherein said gene disruption
comprises an insertion..Iaddend.
Description
This application is a 371 of PCT/FR93/01202 Dec. 7, 1993.
The present invention relates to cells modified with respect to the
catabolism of betaine, to their preparation and their use,
especially for the improved production of metabolites and/or
enzymes. The invention also relates to DNA fragments carrying genes
for the catabolism of betaine.
Glycine betaine (N,N,N-trimethylglycine) is generally known for its
osmoprotective properties, which confer on bacteria tolerance to
osmotic stress (Csonka, 1989). To explain the origin of this
property, it has been proposed that the molecular effects of
glycine betaine on the activity of water and on the osmotic
pressure of the cytoplasm in Escherichia coli were more important
than those of the solutes which it replaces (Cayley et al, 1992).
Furthermore, in addition to its osmoprotective potentials, it has
been described that glycine betaine could also promote the
production of enzymes (JP 8,260,709) or of metabolites, such as
amino acids (Patent JP 202703); antibiotics (Patent AU 825513) and
vitamins (White et al, 1971). However, most bacteria except
cyanobacteria and other CO.sub.2 -fixing prokaryotes do not
synthesize glycine betaine which is mainly synthesized by plants.
It should therefore be added to production media in fermenters,
which generates an additional cost in an industrial process. The
present invention provides a solution to this problem.
The applicant has indeed demonstrated that it is possible, by
modifying the catabolism of betaine of cells, especially by genetic
means, to potentiate the effect of this compound on the production
of enzymes or of metabolites without affecting the rate of growth
of cells, their viability and the like, under industrial
fermentation conditions. The applicant has also identified,
isolated and characterized DNA fragments containing genes involved
in the catabolism of betaine, which make it possible in particular
to prepare cells specifically modified with respect to the
catabolism of betaine, and whose modifications are segregationally
stable and nonreversible. These fragments also make it possible to
stimulate the catabolism of betaine by amplification of the
appropriate enzymatic activities. The present invention therefore
makes it possible to potentiate the effects of betaine and,
thereby, to use this compound economically in industrial
fermentation processes.
A first subject of the invention therefore relates to a modified
cell exhibiting at least one modification with respect to a gene
involved in the catabolism of betaine.
In a first embodiment, the term modified cell designates more
particularly any cell having a substitution and/or a deletion
and/or an insertion of one or more bases in the considered gene(s)
and degrading betaine less rapidly. Such modifications can be
obtained in vitro (on isolated DNA fragments carrying genes for the
catabolism of betaine) or in situ, for example, by means of genetic
engineering techniques, or alternatively by exposing the said cells
to a treatment by means of mutagenic agents.
As mutagenic agents, there may be mentioned for example physical
agents such as energetic radiation (X-, g- or ultraviolet rays end
the like) or chemical agents capable of reacting with various
functional groups of the bases of DNA, and for example alkylating
agents [ethyl methane-sulphonate (EMS),
N-methyl-N'-nitro-N-nitrosoguanidine, N-nitroquinoline 1-oxide
(NQO)], dialkylating agents, intercalating agents and the like.
Deletion is understood to mean the removal of all or part of the
gene considered. This may especially be a portion of the coding
region and/or of all or part of the promoter region for
transcription.
The genetic modifications can also be obtained by gene disruption,
for example according to the procedure initially described by
Rothstein (1983). In this case, all or part of the gene is
preferably perturbed so as to allow the replacement, by homologous
recombination, of the wild-type genomic sequence by a nonfunctional
or mutant sequence prepared in vitro.
The said modification(s) may be located in the coding portion of
the gene or in the regions responsible for the expression end/or
transcriptional regulation of the said genes. The (total or
partial) incapacity of the said cells to degrade betaine can
manifest itself either by the production of inactive enzymes
because of structural or conformational modifications, or by the
absence of production, or by the production of enzymes having an
impaired activity, or alternatively by the production of natural
enzymes at en attenuated level or according to a desired mode of
regulation.
Moreover, certain modifications such as point mutations are by
nature capable of being corrected or attenuated by cellular
mechanisms, for example during the replication of DNA preceding
cell division. Such genetic modifications are, in this case, of
limited interest at the industrial level since the phenotypic
properties resulting therefrom are not perfectly stable. According
to the present invention, it is now possible, by virtue of the
identification of DNA fragments carrying genes for the catabolism
of betaine, to prepare modified cells in which the said
modification(s) are segregationally stable and/or nonreversible.
The cells exhibiting such modifications are particularly
advantageous as cellular host for the production of metabolites
end/or enzymes.
In another particular embodiment, the modified cells of the
invention are cells in which at least one gene involved in the
catabolism of betaine is amplified, and which as a result degrade
betaine more rapidly.
The amplification can be obtained by introducing a DNA fragment
carrying a gene for the catabolism of betaine into the cell. This
fragment is preferably part of a vector, which my be an
autonomously replicating vector or an integrative vector. Moreover,
the DNA fragment may be homologous or heterologous in relation to
the modified cell, that is to say that the amplified gene(s) may be
genes from the said cell or genes obtained from other cellular
sources and encoding an activity of the same type. The choice of
the vector and of the origin of the amplified fragment depends on
the cells considered and the applications envisaged. The DNA
fragment may be introduced into the cells by any method allowing
the introduction of a foreign DNA into a cell. This may be in
particular transformation, electroporation, conjugation, protoplast
fusion, or any other techniques known to persons skilled in the
art.
According to studies carried out essentially in Rizobium meliloti,
the degradation of glycine betaine is performed on media of low
osmolarity, by three successive demethylations (see FIG. 1). The
first stage is catalysed by betaine homocysteine methyltransferase
E.C. 2.1.1.5. and leads to dimethylglycine; the second is catalysed
by dimethylglycine dehydrogenase E.C. 1.5.99.2 and generates
momomethylglycine or sarcosine; finally the third is catalysed by
sarcosine dehydrogenase E.C. 1.5.99.1 and the product of the
reaction is glycine (Smith et al, 1988).
Preferably, the modifications exhibited by the cells of the
invention affect one of the fist two stages of the catabolism of
betaine, or optionally the two first stages simultaneously.
Preferably, the cells of the invention are cells which product
metabolites and/or enzymes. In this respect, they may also be
recombinant cells which product metabolites and/or enzymes, that is
to say cells modified by recombinant DNA techniques so as to
improve their production capacity (cf. especially WO 91/11518, EP
346000). Still more preferably, the cells of the invention are
chosen from cells of the genus Pseudomonas, Streptomyces,
actinomycetes, Propionibacterium, Corynabacterium, Bacillus,
Escherichia, Salmonella, Rhizobium, Agrobacterium,
Rhodopseudomonas, Xanthomonas, Clostridium end
Methanobacterium.
Another aspect of the invention relates to a process for preparing
cells exhibiting a modification of at least one gene involved in
the catabolism of betaine which are capable of being used under
industrial fermentation conditions.
The present invention in effect describes the identification,
isolation and characterization of DNA fragments containing genes
involved in the catabolism of betaine. These fragments now make it
possible to prepare cells which are specifically modified with
respect to the catabolism of betaine. It is indeed possible to
modify in vitro the fragments described so as to make them
nonfunctional and to reintroduce them into a given cell, in which
they will become substituted by double homologous recombination for
the corresponding functional genomic copy. It is also possible, on
the basis of the fragments thus isolated, to prepare probes which
will become integrated into the genome of a desired cell,
specifically in the corresponding gene.
More particularly, the process of the invention consists in
replacing the considered chromosomal gene(s) by versions modified
in vitro.
Another subject of the invention relates to a DNA fragment carrying
at least one gene for the catabolism of betaine.
More particularly, the present invention is illustrated by the
isolation and characterization of DNA fragments from Pseudomonas
denitrificans which complement the mutants blocked in the
catabolism of glycine betaine into dimethylglycine and those
blocked in the catabolism of dimethylglycine into sarcosine, that
is to say the DNA fragments containing genes involved in the
degradation of glycine betaine into dimethylglycine and of
dimethylglycine into sarcosine. The DNA fragments according to the
invention were isolated from a Pseudomonas denitrificans strain
SC510, which is derived from the strain MB580 (U.S. Pat. No.
3,018,225). These fragments were obtained (i) preparation of
mutants blocked in the catabolism of betaine. Various techniques
already mentioned above can be used to this end. The selection of
the is performed by culturing on an appropriate medium and
assaying, according to conventional techniques for persons skilled
in the art, betaine or products of its acid from a microorganism
capable of catabolizing catabolism. (ii) complementation of three
mutants with nucleic betaine, (iii) selection of the complemented
mutants, then isolation and characterization of the nucleic acid
having allowed this complementation, which therefore carries genes
for the catabolism of betaine.
It is clear that, from the DNA fragments identified and isolated in
the present application, persons skilled in the art can, especially
by hybridization experiments, isolate and clone genes for the
catabolism of betaine from other cellular sources.
More preferably, the gene in question is therefore the one encoding
betaine-homocysteine methyltransferase, on which gene a
modification according to the invention induces, in the cell, a
decrease in the betaine-homocysteine methyltransferase activity.
Preferably still, the gene in question is the one encoding
dimethylglycine dehydrogenase, on which a modification according to
the invention induces, in the cell, a decrease in the
dimethylglycine dehydrogenase activity.
Another subject of the invention relates to an improved process for
producing metabolites or enzymes by culturing a cell which produces
the said metabolite or enzyme, modified with respect to its
catabolism of betaine, under production conditions, and then
recovering the said metabolite or enzyme.
According to a first embodiment, the producing cell exhibits at
least one modification with respect to a gene involved in the
catabolism of betaine and degrades betaine less rapidly.
Preferably, according to the first embodiment, the modification(s)
are segregationally stable and/or nonreversible.
In a preferred variant of the invention, the modification(s) are
mutational deletions and/or insertions.
According to a second embodiment, at least one gene of the
producing cell, which is involved in the catabolism of betaine, is
amplified and the said cell degrades betaine more rapidly.
The process of the invention is particularly suitable for the
production of metabolites such as amino acids, vitamins,
antibiotics, their derivatives or their precursors.
The process of the invention can especially allow the improved
production of cobalamin, and preferably of vitamin B12.
The present invention is supplemented by the following examples
which should be considered as illustrative and nonlimiting.
GENERAL MOLECULAR BIOLOGY TECHNIQUES
The methods conventionally used in molecular biology such as
preparative plasmid DNA extractions, plasmid DNA centrifugation in
a caesium chloride gradient, electrophoresis on agarose or
acrylamide gels, purification of DNA fragments by electroelution,
phenol or phenol-chloroform extraction of proteins, DNA
precipitation in salt medium with ethanol or isopropanol,.
transformation in Escherichia coli and the like, are well known to
persons skilled in the art and are widely described in the
literature [Maniatis T. et al., "Molecular Cloning, a Laboratory
Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
1982; Ausubel F. M. et al. (eds), "Current Protocols in Molecular
Biology", John Wiley & Sons, New York, 1987].
The restriction enzymes were produced by New England Biolabs
(Biolabs), or Pharmacia and are used according to the
recommendations of the suppliers.
The pBR322 and pUC type plasmids are of commercial origin (Bethesda
Research Laboratories).
For the ligations, the DNA fragments are separated according to
their size by electrophoresis on agarose or acrylamide gels,
exctracted with phenol or with a phenol/chloroform mixture,
precipitated with ethanol and then incubated in the presence of T4
phage DNA ligase (Boehringer) according to the recommendations of
the supplier.
The filling of the protruding 5' ends is performed by the Klenow
fragment of DNA polymerase I of E. coli according to the
specifications of the supplier. The destruction of the protruding
3' ends is performed in the presence of T4 phase DNA polymerase
used according to the recommendations of the manufacturer. The
destruction of the protruding 5' ends is performed by a controlled
treatment with S1 nuclease.
Site-directed mutagenesis in vitro by synthetic
oligodeoxynucleotides is performed according to the method
developed by Taylor et al. [Nucleic Acids Res. 13 (1985)
8749-8764].
The enzymatic amplification of the DNA fragments by the so-called
PCR technique [Polymerase-catalysed Chain Reaction, Saiki R. K. et
al., Science 230 (1985) 1350-1354; Mullis K. B. et Faloona F. A.,
Meth. Enzyme. 155 (1987) 335-350] is performed using a "DNA thermal
cycler" (Perkin Elmer Cetus) according to the specifications of the
manufacturer.
The verification of the nucleotide sequences is performed by the
method developed by Sanger et al. [Proc. Natl. Acad. Sci. U.S.A.,
74 (1977) 5463-5467].
ABBREVIATIONS
Betaine: glycine betaine or N,N,N-trimethylglycine
HPLC: high-pressure liquid chromatography
DMG: dimethylglycine
kb: kilobase
LEGEND TO THE FIGURES
FIG. 1: Pathway for the degradation of clycine betaine into
glycine. Three successive demethylations take place, the glycine
betaine is catabolized into dimethylglycine which is degraded into
sarcosine which it itself degraded into glycine.
FIG. 2: Growth curve in minimum M medium. The nonmutated SBL27
Rif.sup.r strain and the mutants G3728 and G3900 are cultured in
minimum M medium in the presence of glycine betaine as carbon
source, their growth is represented on the Y-axis as number of
cells per ml, as a function of the time in days described on the
x-axis.
FIG. 3: Mapping of the DNA fragment from P. denitrificans
complementing the mutants G3728, G3736, G3899 and G3900. The
restriction sites and the position of insertion of the transposon
of the corresponding mutants G3728, 33899 end G3900 are indicated
on the DNA fragment. The inserts of the plasmids pXL2100, pXL2101,
pXL2105 and pXL2107 which were used to complement the mutants are
represented below the fragment. + complementation, - no
complementation.
FIG. 4: Mapping of the DNA fragment from P. denitrificans
complementing the mutants G3727 and G3738. The restriction sites
and the position of insertion of the transposon G3738 are indicated
on the DNA fragment. Part of the inserts of the plasmids pXL19E2,
pXL17A10, end pXL13C5 which were used to complement the mutants are
shown below the fragment. + complementation, + no
complementation.
EXAMPLE 1
Isolation of Pseudomonas denitrificans mutants blocked in the
catabolism of glycine betaine.
This example describes the isolation of Pseudomonas denitrificans
mutants blocked in the catabolism of glycine betaine into
dimethylglycine and sarcosine. These mutants were isolated from a
library of mutants, in which the transposon Tn5Sp.sup.r is inserted
into the genome of the Pseudomonas denitrificans strain SBL27
Rif.sup.r. This library was prepared in the following manner: a
plasmid, designated pRK2013::Tn5Sp.sup.r, was constructed by
inserting the spectinomycin resistance gene Sp.sup.r derived from
the plasmid pHP45.OMEGA. (Prentki et al., 1984) into the BamHI site
of the transposon Tn5 (Berg et al., 1983) cloned into the plasmid
pRK2013:: Tn5 (Ditta et al., 1980). A conjugation was then
performed by mixing the exponential phase cultures of
SBL27Rif.sup.r and E. coli MC1060 (pRK1013::Tn5Sp.sup.r). The
transconjugates Rif.sup.r Sp.sup.r were obtained after incubation
at 30.degree. C. for 5 days, at the frequency of 10.sup.-8 clones
per recipient cell. It was checked for 12 clones that the plasmid
introduced had indeed been lost (the plasmid DNA was prepared end
then introduced into E. coli by transformation; no clone carrying
the resistance of the plasmid was obtained) and that the transposon
Tn5Sp.sup.r was indeed integrated into the genome of SBL27Rif.sup.r
(by southern as described in Example 2).
About 3000 mutants of this library were reisolated on solid (15 g/l
of Difco agar) minimum M medium (1 g/l NH.sub.4 Cl, 7 g/l Na.sub.2
HPO.sub.4, 3 g/l KH.sub.4 PO.sub.2 PO.sub.4, 0.5 g/l NaCl, 1 mM
MgSO.sub.4, 0.1 mM CaCl.sub.2, 10 g/l thiamine) where the carbon
source is glycine betaine at 10 g/l and then incubated at
30.degree. C. for 3 days. Next, the betaine, the dimethylglycine
end the sarcosine were assayed in the culture supernatant after
separation Shodex Ionpak S 801 P, length 50 cm, diameter 8 mm;
temperature: 70.degree. C.; mobile phase: 0.01M sodium azide) and
detection by differential refractometry.
Eighteen mutants were found no longer to use betaine as carbon
source, they are 33727 to G3731, G3733 to G3742 and G3897, G3899
and G3900.
Moreover, when these mutants are cultured, according to the
procedure already described (Cameron et al, 1989), in 10 ml of PS4
medium for 5 days at 30.degree. C., betaine, or dimethylglycine or
sarcosine can be measured in the culture supernatant after
separation by HPLC end detection by differential refractometry.
Betaine is detected in the broth supernatant of the mutants G3728,
G3736, G3897, G3899 and G3900 (between 53 and 98 % of the betaine
contained in the initial medium), whereas with the mutants G3727,
G3738 and G3742, it is dimethylglycine (about 88 to 97% of the
betaine contained in the initial medium); with the mutants G3729,
G3730, G3731, G3733, G3737 and G3741, it is sarcosine (about 64 and
75% of the betaine introduced into the medium); with the other
mutants G3734, G3735, G3739, G3740 and with the nonmutated strain,
none of the three products is observed, see Table 1.
Furthermore, only SBL27 Rif.sup.r and the mutants G3728, G3736,
G3897, G3899 and G3900 grow on solid minimum M medium where the
carbon source is dimethylglycine (10 g/l). Cultures in liquid
minimum M medium where the carbon source is glycine betaine at 10
g/l are performed as follows: starting with a reisolation on rich
LB medium (Cameron et al, 1989), the strain is cultured in 5 ml of
LB medium at 30.degree. C. for 12 hours; the cells of this
preculture are washed in minimum M medium, then inocula of 0.2% are
inoculated, in eight Erlenmeyer flasks of 250 ml containing 25 ml
of minimum M medium where the carbon source is glycine betaine (10
g/l), and incubated with stirring at 250 rpm, at 30.degree. C. On
days 3, 4, 5, 6, 10, 14, 21, the cells contained in an Erlenmeyer
flask are counted after plating and growth on solid minimum medium
where the carbon source is glycine betaine. When the mutants G3728
and G3900 are cultured in liquid minimum medium where the carbon
source is glycine betaine (10 g/l), for the first three days, no
growth is observed whereas with the strain SBL27 Rif.sup.r the
stationary phase is already reached, see FIG. 2; however, on the
sixth day, a stationary phase comparable to that of SBL27 Rif.sup.r
is observed. This result shows that the mutants which do not
degrade all the betaine (10 g/l) contained in the PS4 medium, see
Table 1, are capable of using it, but after a lag phase of 3 days
compared with the no-mutated strain, as carbon source in a minimum
medium containing 10 g/l of betaine.
These physiological data make it possible to demonstrate in
Pseudomonas denitrificans SBL27 Rif.sup.r a pathway for the
degradation of glycine betaine into dimethylglycine and sarcosine
where: 1) the stage for the degradation of glycine betaine into
dimethylglycine is blocked (or partially blocked) in the mutants
G3728, G3736, G3897, G3899 and G3900; 2) the stage for the
degradation of dimethylglycine into sarcosine is blocked in the
mutants G3727, G3738 and G3742; 3) the stage for the degradation of
sarcosine is blocked in the mutants G3729, G3730, G3731, G3733,
G3737 and G3741.
EXAMPLE 2
Genetic characterization of the Pseudomonas denitrificans mutants
blocked in the catabolism of betaine.
This example describes the molecular biology experiments which make
it possible to classify and characterize the mutants with the aid
of their transposon. The genotype of each mutant is analysed by
Southern (Maniatis et al, 1982) in the following manner: the
genomic DNA of each mutant (G3727 to G3731, G3733 to G3742 and
G3897, G3899 and G3900) was prepared and then all aliquots is
digested with the restriction enzyme EcoRI; the fragments thus
obtained are separated by electrophoresis on an agarose gel and are
then transferred onto a Biodyne membrane; this membrane is then
hybridized with one of the following plasmids labelled with
.alpha..sup.32 P-dCTP. These plasmids are pT27, pT28, pT30, pT31,
pT34, pT35, pT36, pT37, pT38, pT39, pT40, pT42, pT97, pT00. These
plasmids were obtained by insertion at the EcoRI site of the vector
pRK290 of the unique genomic EcoRI fragment in which is inserted
the transposon Tn5Sp.sup.r of the mutants G3727, G3728, G3730,
G3731, G3734, G3735, G3736, G3737, G3738, G3739, G3740, G3742,
G3897, G3900 respectively. The plasmids pT carrying the desired
EcoRI fragment are selected by their resistance to spectinomycin,
conferred by the transposon. On hybridizing the membrane for
example with the plasmid pT27, the genomic DNA of the mutants G3727
and G3738 shows only one radioactive band corresponding to an EcoRI
fragment with a molecular weight of about 16 kb, which indicates
that, in these mutants, the transposon is inserted at the same
position. In contrast, for the nonmutated strain, the hybridizing
fragment is 8 kb and with all the other mutants two fragments
hybridize, one comigrates with that of the non-mutated strain and
the other has a variable size greater than the size of the
transposon (8 kb) (de Bruijn, 1987; Prentki, 1984). The successive
hybridizations with each of the plasmids made it possible to group
the mutants by classes of hybridization of 1 to 12, see Table
1.
Moreover, the plasmids pT27, pT28, pT30, pT31, pT34, pT36, pT37,
pT38, pT97, pT00 were used to reintroduce by double homologous
recombination the mutation derived from the insertion of the
transposon, into the nonmutated strain SBL27 Rif.sup.r, according
to a procedure already described (Cameron et al, 1991). For the
strains thus obtained by gene disruption, the genotype was checked
by Southern as has Just been described in the preceding paragraph,
and the phenotype was determined after analysis of the compounds
detected in the supernatant of the cultures in PS4 medium as
described in Example 1.
For all the strains obtained by gene disruption, which lead to the
same phenotype as the initial mutated strains, see Table 1, the
quantities of the compounds detected are comparable. These strains
belong: 1) either to the hybridization class 5 and accumulate
betaine, 2) or to the hybridication class 2 and accumulate
dimethylglycine, 3) or to the hybridization class 4 and accumulate
sarcosine.
Furthermore, a deletion of a BgIII genomic fragment of 10 kb and
insertion of a spectinomycin resistance cassette were carried out
using the plasmid pT28. The strain, whose genotype was checked by
Southern (as described in the first paragraph of this example) has
the same betaine accumulation phenotype as the strains described in
the hybridization class 5.
TABLE 1 Clarification of the mutants blocked in the catabolism of
betaine into sarcosine. Strains Compounds detected in the strains
No. Hybridization initial genetic G classes mutants disruptants
3727 2 DMG 88% DMG 3728 5 Betaine 53% Betaine sarcosine 13% 3729 3
Sarcosine 68% ND 3730 3 Sarcosine 69% 0 3731 4 Sarcosine 64%
Sarcosine 3733 4 Sarcosine 69% ND 3734 7 0 0 3735 8 0 ND 3736 1
Betaine 62% 0 3737 4 Sarcosine 75% Sarcosine 3738 2 DMG 91% DMG
3739 9 0 ND 3740 10 0 ND 3741 4 Sarcosine 64% ND 3742 11 DMG 97% ND
3897 12 Betaine 96% 0 3899 5 Betaine 95% ND 3900 5 Betaine 98%
Betaine
EXAMPLE 3
Complementation of Pseudomonas denitrificans mutants blocked in the
stages for demethylations of glycine betaine into sarcosine.
This example describes the isolation of DNA fragments from P.
dentrificans carrying genes involved in the degradation of betaine
into sarcosine. These fragments were detected by hybridization
experiments, using the insert of the plasmids pT28 or pT38 as probe
and the library of plasmids containing Sau3AI fragments of the P.
denitrificans DNA which are cloned into pXL59 (Cameron et al,
1989). The inserts of the plasmids hybridizing with the probe were
mapped. Clones and subclones, which were constructed (Maniatis et
al, 1982) in the vectors derived from RSF1010 (Cameron et al,
1989), were introduced by conjugation (Cameron et al, 1989) in the
mutants blocked in the catabolism of betaine into dimethylglycine
or in the mutants blocked in the degradation of dimethylglycine
into sarcosine. The complementation of the mutants by the clones or
the subclones was determined by the absence of accumulation of
betaine, dimethylglycine or sarcosine in the culture supernatant of
the transconjugant strains cultured in PS4 medium, as described in
Example 1. 3-1 DNA fragment from P. denitrificans carrying genes
involved in the degradation of betaine into dimethylglycine.
The clones hybridizing with pT28, were introduced into the mutants
G3728, G3736, G3897, G3899 and G3900 blocked in the catabolism of
betaine into dimethylglycine. As is presented in FIG. 3, two
subclones pXL2105 and pXL2107 contained in the EcoRI fragment of 12
kb complement four mutants among the five G3728, G3736, G3899 and
G3900. One of the subclones pXL2105 contains a 3.4 kb SstI-XhoI
fragment cloned into the vector pXL435 (Cameron et al, 1989) and
complements two mutants G3900 and G3899; the other pXL2107 contains
a 4 kb BamHI-EcoRI fragment cloned into pKT230 (Cameron et al,
1989) and complements the mutants G3728 and G3736. Moreover, the
insertion of the transposon in the mutants G3728, G3899 and G3900
has indeed beam mapped on this 12 kb EcoRI fragment, which is not
observed in the mutant G3736. It is possible that the phenotype of
this mutant is not correlated with the position of the transposon
since the mutant obtained after reverse genetics does not
accumulate betaine, see Example 2. Consequently, at least two genes
are involved in the stage for degradation of betaine into
dimethylglycine. 3-2 DNA fragment from P. denitrificans carrying
genes involved in the degradation of dimethylglycine into
sarcosine.
The clones hybridizing with pT38 were introduced into the mutants
G3727, G3738 blocked in the catabolism of dimethylglycine into
sarcosine. As is presented in FIG. 4, two clones derived from the
library and mapping on a 14.5 kb EcoRI-EcoRI-EcoRI fragment
complements G3727 and 33738. One of the subclones pXL19E2 contains
a 6.6 kb EcoRI fragment and the other pXL13C5 contains the 14.5 kb
EcoRI-EcoRI-EcoRI fragment. Consequently, at least one gene, mapped
on the fragment described in FIG. 4, is involved in the stage for
degradation of dimethylglycine into sarcosine.
EXAMPLE 4
Improvement of the production of vitamin B.sub.12 in a Pseudomonas
denitrificans strain modified in the catabolism of glycine
betaine.
This example illustrates the improvement in the production of
cobalamins in a cobalamin-producing strain by disruption of genes
involved in the catabolism of betaine into dimethylglycine.
The mutation, which is responsible for the phenotype of the mutant
G3728 (or G3900), is generated by the transposon which is mapped on
the EcoRI fragment, cloned into pT28 (or pT00) and described in
Example 2. This mutation was introduced by double homologous
recombination into the cobalamin-producing P. denitrificans strain
SC510 Rif.sup.r (Cameron et al, 1991). For both strains thus
obtained SC510 Rif.sup.r ::pT28 and SC510 Rif.sup.r ::pT00 by
reverse genetics, the genotype was checked by Southern as described
in Example 2, and the production of cobalamins was determined after
analysis by HPLC (column: Asahibak OD50, length: 15 cm, diameter: 6
mm, mobile phase: acetonitrile/0.1M potassium cyanide) and
ultraviolet detection (wavelength: 365 nm) of the vitamin B.sub.12
contained in cultures of the strains grown for seven days at
30.degree. C. in 250 ml Erlenmeyer flasks containing 25 ml of PS4
medium where the betaine glycine is at 2 mg/l, according to a
procedure already described (Cameron et al, 1989). The results of
these assays are indicated in Table 2. They represent the mean
value obtained for two cultures in the four experiments performed
independently.
Under the culture conditions used, the production of cobalamins is
increased by a factor of 10, compared with the SC510Rif.sup.r
strain, in the mutated strains in an EcoRI genomic fragment of 13
kb, see FIG. 3, whose DNA encodes genes involved in the catabolism
of betaine into dimethylglycine. These results show clearly that by
modifying the catabolism of the betaine of the strains of the
invention, their metabolite-producing capacity has been increased.
Table 2. Production of vitamin B.sub.12 in the strains mutated in
the EcoRI fragment carrying genes involved in the catabolism of
betaine into dimethylglycine. The levels of production of the
modified strains are given relative to the production of the Sc510
Rif.sup.r strain, which was arbitrarily set at 1.
Strains/experiment 1 2 3 4 Sc510 Rif' 1 1 1 1 SC510 RifnpT28 10.5
11 22 14.5 SC510 RifnPT00 11 8
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