WO2001021819A2 - Method for genetic modification of lactobacillus delbrueckii - Google Patents
Method for genetic modification of lactobacillus delbrueckii Download PDFInfo
- Publication number
- WO2001021819A2 WO2001021819A2 PCT/FR2000/002565 FR0002565W WO0121819A2 WO 2001021819 A2 WO2001021819 A2 WO 2001021819A2 FR 0002565 W FR0002565 W FR 0002565W WO 0121819 A2 WO0121819 A2 WO 0121819A2
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- WO
- WIPO (PCT)
- Prior art keywords
- plasmid
- bacteria
- chromosome
- delbrueckii
- vector
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
Definitions
- the present invention relates to the genetic modification of Lactobacillus delbrueckii.
- Lactic acid bacteria are widely used in the food industry, particularly for the manufacture of various fermented products; moreover, their harmlessness made recommend their use for the production by genetic engineering of various substances intended in particular for a therapeutic use.
- lactic acid bacteria makes it possible to adapt their characteristics according to the intended use, whether by the introduction and expression of foreign DNA, or by overexpression or, on the contrary, the inactivation of genes naturally present in said bacteria.
- bacteria For a modification to be stable, it must be integrated into the bacterial chromosome.
- the desired modification is inserted into a non-replicating plasmid carrying a selection marker.
- the vector thus obtained is introduced into the bacteria; the bacteria expressing the selection marker are then recovered, which are those which have integrated the vector into their chromosome.
- the modification results from 2 low frequency events: 1) the introduction of the plasmid into the bacteria; 2) integration of said plasmid into the chromosome.
- Lactobacillus delbrueckii which notably comprises the subspecies Lb. delbrueckii subsp. bulgaricus, Lb. delbrueckii subsp. lactis, and Lb. delbrueckii subsp. delbrueckii.
- delbrueckii is described in the prior art: it is the conjugative plasmid pAM ⁇ 1, described as non-replicative in Lb. delbrueckii.
- Application EP 0603416 in the name of MEIJI MILK PRODUCTS CO LTD reports the use of this plasmid to integrate a modification into the Lb chromosome.
- delbrueckii. a selection marker (resistance to erythromycin) has been inserted into a fragment homologous to a region of the Lb chromosome. delbrueckii, and the whole was introduced into the plasmid pAM ⁇ 1.
- the integrative plasmid thus constructed was multiplied in Lactococcus lactis, then transferred by conjugation to Lb. delbrueckii, - the transconjugants / integrants are then selected on the basis of the resistance to erythromycin resulting from the integration, by homologous recombination, of the insert of the plasmid into the chromosomal DNA.
- conditional plasmids as vectors.
- a vector for example a plasmid, of which at least one of the functions of replication, of partition, or of stability is only active under certain conditions (for example temperature, pH, concentration of mineral salts, presence in the culture medium of a particular compound, etc.) or the fact that one or more of the proteins involved in this function contain (s) one or more mutations making the activity conditional, either because one or more of the proteins involved in this function are placed under the control of an inducible promoter.
- the modification can thus be carried out in two stages: in the first, the plasmid is introduced at permissive temperature into the bacteria, which allows a first selection of those in which it is established; in the second, these bacteria are cultivated at non-permissive temperature for the replication of the plasmid, which makes it possible to select those in which the introduced sequences are integrated into the chromosome.
- PCT Application WO 93/18164 describes the use of these plasmids in various species of lactic acid bacteria, including Lb. delbruckii subsp. bulgaricus.
- the subject of the present invention is therefore the use of a plasmid comprising the theta replication system of pIP501 or a related replication system, as a conditional, heat-sensitive, integration vector enabling a modification of the genetic information of a bacteria of the species Lb. delbrueckii. This modification is introduced into the chromosome via one or more homologous transposition and / or recombination events.
- replication system related to the theta replication system of pIP501 any theta replication system having the functional characteristics, and in particular the thermosensitivity in Lb delbrueckii, of pIP501.
- the percentage identity of a sequence with a reference sequence is defined here as the percentage of residues of this sequence which are identical with those of the reference sequence on an alignment of the 2 sequences ensuring maximum correspondence between the positions of the residues .
- the present invention relates in particular to a method for modifying the genetic information carried by the chromosome of a bacterium of the species Lb delbrueckii, characterized in that it comprises: a) the construction of an integrative plasmid, by insertion of at least one DNA sequence capable of integrating into the bacterial chromosome, into a conditional vector comprising the thIP replication system of pIP501 or a related replication system, said vector further carrying at least one selection marker ; b) the introduction of the plasmid into said bacterium and the multiplication of the latter, in permissive conditions for the replication and the maintenance in stable form of said plasmid; c) the multiplication of bacteria expressing at least one plasmid selection marker at the end of step b), under non-permissive conditions for replication and / or maintenance in stable form of the plasmid; and optionally, d) recovering the bacteria expressing at least one selection marker originating from the plasmid, at the end of step c).
- the step of multiplying the bacteria under permissive conditions can be omitted for the replication and the maintenance of the plasmid in stable form; in this case, after the introduction of the plasmid into the bacterium, the multiplication of the latter is carried out directly under non-permissive conditions for the replication and / or maintenance in stable form of the plasmid.
- the method according to the invention further comprises the following steps: e) excision of the sequences originating from the vector, by multiplication of the bacteria expressing, at the end of step c), at least one marker for selection from the plasmid, and advantageously, f) elimination of the excised DNA, by multiplication of the bacteria obtained in step e) under non-permissive conditions for replication and maintenance of the vector in stable plasmid form.
- Conditional vectors including the thIP replication system of pIP501, or a related system, used in Lb. delbrueckii for setting work of the present invention are thermosensitive plasmids capable of replicating and maintaining themselves at 35-37 ° C in this bacterial species, and whose replication and / or maintenance in stable form are inhibited from approximately 42 ° C.
- These plasmids also carry at least one selection marker, namely a gene which can be expressed in Lb. delbrueckii and whose expression confers on the bacteria harboring a distinctive phenotype allowing their selection. It may be, for example, a resistance marker, conferring a character of resistance to a substance usually toxic to the bacterium, for example an antibiotic, or else an auxotrophy marker conferring the ability to grow in it. lack of a nutrient usually essential for the bacteria. The bacteria expressing these markers are for example easily selected by their survival on a selective medium, namely in the presence of said toxic substance or in the absence of said nutrient.
- the bacteria in which the plasmid is present can be selected on the basis of the expression of a selection marker originating from the plasmid.
- the bacteria in which the DNA of the plasmid has been integrated into the chromosome can be selected on the basis of the expression of a selection marker coming from the plasmid constructed at 1 'step a).
- Another possibility of selection at the end of step c) consists in using a property of the strain which results from the integration of the plasmid and / or from the excision of the vector sequences.
- conditional plasmid comprising a selection marker M (for example a resistance marker), and a DNA sequence capable of integrating by homologous recombination into a bacterial gene X, said gene X being essential under certain conditions for the bacterium (for example a gene essential for its growth on a given medium), the integration of the plasmid inactivating the gene X, and the excision of the sequences of the vector restoring its activity.
- a selection marker M for example a resistance marker
- the bacteria in which the plasmid DNA has been integrated can be selected on the basis of the expression of the selection marker M, under conditions in which the X gene is not essential, and the bacteria in which the sequences of the integrated vector have been excised can be selected, under conditions in which the X gene is essential, on the basis of the restoration of its activity.
- This selection mode also also makes it possible to select, in a single step, bacteria in which the integration of the plasmid DNA and the excision of the vector sequences have taken place; in this case, the bacteria are cultured directly under conditions in which the X gene is essential, and which are also selective for the M marker.
- the bacteria selected will be those in which the integration of the plasmid and the excision of the vector sequences (including including marker M) restored the activity of the X gene.
- DNA sequences capable of integrating into the chromosome of a bacteria of the species capable of integrating into the chromosome of a bacteria of the species
- Lb. delbrueckii include in particular: sequences chosen on the basis of their homology with a portion of the chromosome where one wishes to introduce a modification, that is to say having sufficient homology with this portion of the chromosome to be able to recombine with it; transposable sequences, in particular transposons or insertion sequences (IS); such sequences can to be inserted at random or with a certain specificity in the bacterial chromosome.
- the modifications which it is wished to integrate into the bacterial chromosome can be made by the simple integration of these sequences, which can for example lead to the inactivation of a gene inside which it takes place. It is also possible to use these sequences to integrate a DNA fragment, in particular a gene of interest of heterologous origin, or a DNA fragment of Lb. delbrueckii previously modified, in the bacterial chromosome.
- step a) of the method according to the invention in a conditional vector in Lb. delbrueckii, comprising the thIP replication system of pIP501 or a related system, as defined above. Integration takes place by recombination (simple crossmg-over) between the fragment of cloned chromosomal DNA and the homologous region of the bacterial chromosome.
- sequences which can be integrated by homologous recombination makes it possible in particular: to inactivate one (or more) bacterial gene (s), - to modify the expression of the genes and / or the activity of the coded products by these genes; - to introduce, in a stable way, new functions in the chromosome, - to study and use the expression of genes in si tu; - to mutate the chromosome by integration via random chromosomal fragments;
- the transposable sequence will be inserted into the plasmid; integration into the chromosome makes it possible to carry out the modification, thus to obtain mutants having interesting characteristics and to characterize more easily the mutated gene (s).
- the structure transposed into the chromosome can be made up of the vector sequences framed on either side by a copy of the transposable sequence.
- transposable sequences can come from bacteria belonging to the Lb species. delbrueckii, or come from other bacterial species, especially other lactic acid bacteria. They can be transposons or insertion sequences.
- Functional transposable sequences in Lb. delbrueckii can be identified by inserting a transposable sequence to be tested (transposon or insertion sequence), in a vector with conditional replication in Lb. delbrueckii comprising the thIP replication system of pIP501 or a related system, as defined above, by implementing steps b) and c) of the process according to the invention, and by looking for the presence of transposants in the from these steps.
- the inventors have thus demonstrated 2 functional insertion sequences in Lb. delbrueckii.
- the present invention also relates to the use of one or other of these sequences to modify the Lb chromosome. delbrueckii.
- the present invention also relates to any integrative plasmid resulting from the insertion of one of these 2 sequences into a vector with conditional replication in Lb. delbrueckii, and in particular in a vector comprising the replication system of pVE6002, such as those described in PCT Application WO 93/18164 or in a vector comprising the theta replication system of pIP501 or a related system, as defined above .
- said vector is a non-conjugative vector.
- Integrative plasmids in accordance with the invention are in particular illustrated by the plasmid pVI49 and the plasmid pVI52.
- the excision of the sequences originating from the vector can be carried out, during step e) of the method. according to the invention, by recombination between the homologous sequences flanking the sequences of the vector.
- a second recombination event (double crossing-over) can take place between the homologous regions duplicated on either side of the sequences of the vector. This event leads to the excision of the vector sequences, and makes it possible, for a fraction of the clones, to replace the wild chromosomal form by the modified plasmid form as shown in FIG. 1.
- A, B chromosomal DNA cloned into the vector
- NP non-permissive conditions.
- these also constitute, on either side of the vector sequences, homologous regions favorable to recombination events which lead to the excision of the vector sequences , and one copy of the SI, the other remaining in the bacterial chromosome at the transposition site.
- FIG. 2 represents the excision of the sequences of a vector by homologous recombination between ISs.
- NP non-permissive conditions.
- the bacteria selected at non-permissive temperature during step c) (> 42 ° C), are cultivated without selection pressure to allow the recombination between homologous regions flanking the vector sequences.
- step f) of the method according to the invention After elimination of the excised sequences, in accordance with step f) of the method according to the invention, the following is thus obtained: in the case of a plasmid comprising a sequence capable of integrating by homologous recombination, a bacterium different from the bacterium -Original host by the presence, in its chromosome, of the modification brought about by this sequence; in the case of a plasmid comprising a transposable sequence, a bacterium which differs from the original host bacterium by the presence, in its chromosome, of a copy of said transposable sequence.
- pVI1055 This plasmid, called pVI1055, is shown in FIG. 3. It carries an ery gene for resistance to erythromycin (Ery).
- electroporation buffer 0.4 M sucrose, 1 mM MgCl 2 , 5 mM KH 2 P0 4 , pH 6.0
- the suspension is incubated for 20 min at 45 ° C. and then cooled on ice.
- An 80 ⁇ l aliquot of the suspension is mixed with the plasmid DNA (-1.5 ⁇ g) and the mixture is transferred to a 0.2 cm electroporation tank.
- the electroporation is carried out at 1 kV, 800 ⁇ , and 25 ⁇ F.
- the mixture is diluted in 2 ml of expression medium (0.2 M sucrose, 5% skimmed milk powder, 0.1% yeast extract, 1% casamino acids, 25 mM MgCl 2 ). After incubation for 3 h at 37 ° C.
- the cells are spread on dishes of selective medium (MRS Agar with 10 ⁇ g / ml of erythromycin); the dishes are incubated in an anaerobic jar at 37 ° C. for 48 h, and the colonies resistant to erythromycin are selected.
- MRS Agar with 10 ⁇ g / ml of erythromycin
- thermosensitivity of pGB305 ⁇ and pVI1055 was first evaluated by Lb transformation tests.
- delbrueckii (ATCC 11842 or VI104) according to the protocol described above, with spreading on selective medium at 37 ° C and 42 ° C.
- transformants are obtained at 37 ° C but not at 42 ° C.
- thermosensitivity of pVI1055 was then confirmed by comparison of its stability at 37 ° C and 44 ° C.
- a culture in MRS / Ery (10 ⁇ g / ml) at 37 ° C was diluted in MRS without Ery and then incubated in parallel at 37 ° C and 44 ° C. Samples are taken at different times, and the cells are spread after dilution on MRS and MRS / Ery, in order to determine the proportion of cells which have lost the plasmid.
- Isolated insertion sequences of lactic acid bacteria were cloned into pVI1055.
- the multiplication of a transformant generates a bacterial population containing the plasmid.
- the SI present on the plasmid in the bacterial population can transpose into the chromosome.
- the transposed structure present in the chromosome can correspond to the plasmid vector, framed on both sides by a copy of the SI, as shown in Figure 4.
- non-permissive temperature 44 ° C
- the plasmids containing the ISs lead to obtaining Ery R clones with a frequency higher than that observed for pVI1055, the plasmid without IS.
- the structural analysis of the chromosomal DNA by Southern Blot with a probe corresponding to the IS tested makes it possible to verify that the integration does indeed result from a transposition event. Highlighting the transposition;
- IS 1223 WALKER and KLAENHAMMER, 1994, cited reference
- 1201 TAILLEZ et al., 1994, cited reference
- the strains containing the plasmids carrying the ISs to be tested were cultured at 37 ° C. in MRS / Ery (10 ⁇ g / ml). Then the cells are diluted in MRS and incubated at 44 ° C. At different times, samples are taken and spread on MRS dishes (to determine the count of viable cells) and on MRS / Ery (Ery R cell count).
- IS1223 (pVI48 / pVI49): The plasmids pVI48 or pVI49 were introduced into VI104. In the presence of IS1223, the frequency of Ery R cells is higher than with pVI1055 alone. After digestion, the chromosomal DNAs of the Ery R clones obtained are hybridized with IS1223. In all cases, the Ery R clones result from transposition events.
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002385072A CA2385072A1 (en) | 1999-09-17 | 2000-09-15 | Method for genetic modification of lactobacillus delbrueckii |
EP00964297A EP1212437A2 (en) | 1999-09-17 | 2000-09-15 | Method for genetic modification of lactobacillus delbrueckii |
AU75265/00A AU7526500A (en) | 1999-09-17 | 2000-09-15 | Method for genetic modification of lactobacillus delbrueckii |
NO20021277A NO20021277L (en) | 1999-09-17 | 2002-03-14 | Method of genetic modification of Lactobacillus Delbrueckii |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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FR9911684 | 1999-09-17 | ||
FR99/11684 | 1999-09-17 |
Publications (2)
Publication Number | Publication Date |
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WO2001021819A2 true WO2001021819A2 (en) | 2001-03-29 |
WO2001021819A3 WO2001021819A3 (en) | 2001-08-09 |
Family
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Application Number | Title | Priority Date | Filing Date |
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PCT/FR2000/002565 WO2001021819A2 (en) | 1999-09-17 | 2000-09-15 | Method for genetic modification of lactobacillus delbrueckii |
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EP (1) | EP1212437A2 (en) |
AU (1) | AU7526500A (en) |
CA (1) | CA2385072A1 (en) |
NO (1) | NO20021277L (en) |
WO (1) | WO2001021819A2 (en) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1993018164A1 (en) * | 1992-03-13 | 1993-09-16 | Institut National De La Recherche Agronomique | Thermosensitive plasmid |
EP0603416A1 (en) * | 1992-07-10 | 1994-06-29 | Meiji Milk Products Company Limited | METHOD OF INTEGRATING GENE INTO CHROMOSOME OF $i(LACTOBACILLUS DELBRUECKII) SPECIES AND PRODUCT OF GENE INTEGRATION |
-
2000
- 2000-09-15 WO PCT/FR2000/002565 patent/WO2001021819A2/en not_active Application Discontinuation
- 2000-09-15 CA CA002385072A patent/CA2385072A1/en not_active Abandoned
- 2000-09-15 EP EP00964297A patent/EP1212437A2/en not_active Withdrawn
- 2000-09-15 AU AU75265/00A patent/AU7526500A/en not_active Abandoned
-
2002
- 2002-03-14 NO NO20021277A patent/NO20021277L/en not_active Application Discontinuation
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1993018164A1 (en) * | 1992-03-13 | 1993-09-16 | Institut National De La Recherche Agronomique | Thermosensitive plasmid |
EP0603416A1 (en) * | 1992-07-10 | 1994-06-29 | Meiji Milk Products Company Limited | METHOD OF INTEGRATING GENE INTO CHROMOSOME OF $i(LACTOBACILLUS DELBRUECKII) SPECIES AND PRODUCT OF GENE INTEGRATION |
Non-Patent Citations (2)
Title |
---|
TAILLIEZ PATRICK ET AL: "Characterization of IS1201, an insertion sequence isolated from Lactobacillus helveticus." GENE (AMSTERDAM), vol. 145, no. 1, 1994, pages 75-79, XP002086414 ISSN: 0378-1119 cité dans la demande * |
WALKER D C ET AL: "Isolation of a novel IS3 group insertion element and construction of an integration vector for Lactobacillus spp." JOURNAL OF BACTERIOLOGY, vol. 176, no. 17, 1994, pages 5330-5340, XP000929387 ISSN: 0021-9193 cité dans la demande * |
Also Published As
Publication number | Publication date |
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NO20021277L (en) | 2002-03-15 |
CA2385072A1 (en) | 2001-03-29 |
AU7526500A (en) | 2001-04-24 |
EP1212437A2 (en) | 2002-06-12 |
NO20021277D0 (en) | 2002-03-14 |
WO2001021819A3 (en) | 2001-08-09 |
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