EP0765397A1 - Verfahren zum einfuhren von genetisches material in mikroorganismen und damit hergestellten transformanten - Google Patents

Verfahren zum einfuhren von genetisches material in mikroorganismen und damit hergestellten transformanten

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Publication number
EP0765397A1
EP0765397A1 EP95921168A EP95921168A EP0765397A1 EP 0765397 A1 EP0765397 A1 EP 0765397A1 EP 95921168 A EP95921168 A EP 95921168A EP 95921168 A EP95921168 A EP 95921168A EP 0765397 A1 EP0765397 A1 EP 0765397A1
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EP
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Prior art keywords
microorganism
bifldobacterium
aforegoing
bacteria
nucleic acid
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EP95921168A
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English (en)
French (fr)
Inventor
Robert Jan Leer
Alessandra Argnani
Pieter Hendrik Pouwels
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Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek TNO
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Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek TNO
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Priority to EP95921168A priority Critical patent/EP0765397A1/de
Publication of EP0765397A1 publication Critical patent/EP0765397A1/de
Withdrawn legal-status Critical Current

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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
    • 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
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • 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)

Definitions

  • the invention lies in the field of genetic engineering or modification using recombinant DNA technology and relates in particular to the genetic modification of microorganisms, in particular Lactic Acid Bacteria, more specifically to bacteria of the genus Bifldobacterium.
  • the invention relates to methods for introducing nucleic acid into said microorganisms, to plasmids that can be employed for transforming said microorganisms, to markers that can be used to select for transformed microorganisms, to transformants which are obtainable by the previously mentioned methods and to products that can be obtained through expression of genes present on plasmid vectors in said microorganisms.
  • the invention particularly relates to such methods for introducing nucleic acid into microorganisms which can hardly be transformed or transfected by the methods existing in the state of the art. These microorganisms are often referred to (and will be hereinunder) as "recalcitrants". Some lactic acid bacteria, especially some Lactobacillus and Bifldobacterium species qualify as recalcitrants.
  • the invention specifically relates to methods for introducing nucleic acids into these organisms and to the organisms obtainable thereby.
  • the invention relates to applications of said microorganisms.
  • Bifidobacteria constitute one quarter of the gut flora of normal, healthy adults (H.M. Modler, R.C. McKeller and M. Yaguchi (1990) Can. Inst . Food Sci. Technol. J. 23(1), 29-41) . These bacteria are thought to have beneficial properties for their host.
  • the use of bifidobacteria as starter cultures for the preparation of yoghurt and other fermented milk products thus may help in promoting health.
  • the genera Bifldobacterium and Lactobacillus were recognized for many years as genuine members of the Lactic Acid Bacteria (LAB) group. According to more recent views the two genera are classified in two different families.
  • LAB Gram-positive, anaerobic, micro-aerophilic, catalase-negative rods or cocci (genus Lactococcus) , including the genera Lactobacillus, Lactococcus, Pediococcus, Strepto ⁇ coccus, Leuconostoc, Enterococcus and Carnobacterium; most importantly, they all produce lactic acid as an important end product from the energy yielding fermentation of sugars. Besides the health promoting properties of some Bifldobacterium and Lactobacillus species for humans, the economic importance of these microorganisms is beyond doubt, because they are widely used as starter cultures for the production of feed- and food products, especially dairy products.
  • the present invention relates to the development of a system for the efficient and reproducible genetic transformation of microorganisms, especially of "recalcitrant strains" of bacteria.
  • Recalcitrant bacteria are defined herein as bacterial strains that cannot be transformed, or with great difficulty only, by any of the common transformation protocols used by persons skilled in the art.
  • Commonly used transformation techniques involve the introduction of DNA in bacteria, i) after rendering the bacteria transformation competent, ii) after protoplast formation of the bacteria, or iii) by various electroporation procedures.
  • the present invention relates in particular to a procedure for transforming bacteria of specific genera of LAB, such as for example bacteria of the genus Bifldobacterium, or of specific strains of LAB genera.
  • LAB genus of the genus Bifldobacterium
  • specific strains of LAB genera such as for example bacteria of the genus Bifldobacterium, or of specific strains of LAB genera.
  • lactobacillus species useful transformation procedures are available.
  • some Lactobacillus strains e.g. specific strains of Lactobacillus J ulgaricus, Lactobacillus lactis, Lactobacillus helveticus, Lactobacillus amylovorus etc
  • the invention provides a solution for these species.
  • the present invention provides a novel method for electro- poration of recalcitrant bacteria of the genus Bifldobacterium.
  • the novel method can, in principle, be applied to any bacterial strain that cannot be transformed by any of the commonly used transformation procedures mentioned above.
  • Bifidobacteria are Gram-positive, strictly anaerobic, catalase-negative, fermentative rods, that are often Y- or V- shaped. Traditionally, bifidobacteria are considered as members of the LAB, although this classification is not unanimously accepted. In this report, we will treat bifidobacteria as members of the group of LAB. Bifidobacteria produce acetic acid and lactic acid (3:2) as their major end products. Based on DNA- DNA hybridization measurements and sugar fermentation patterns, 25 species are distinguished within the genus Bifldobacterium in the present classification. The GC-content of bifidobacteria is high, between 55 and 64% (Scardovi, V.
  • Bifidobacteria are among the most abundant species in the lower small intestine of man and animals.
  • the distal ileum may contain 10 5 -10 7 organisms per ml of contents (Gorbach, S.L. et al, (1967) Gastroenterology 53, 856-867; Drasar, B.S. et al, (1969) Gastroenterology 56, 71-79) .
  • bifidobacteria are also prominent, unlike lactobacilli, and are present at concentrations of 10 10 or more per gram contents, constituting 5-10% of total flora in the bowl (Mitsuoka, T. (1992) In: The lactic Acid Bacteria; The Lactic Acid Bacteria in Health and Disease (B.J.B. Wood Ed.) pp 69-114 Elsevier Applied Science) .
  • B .bifidum and B . longum are predominant species in these three habitats, while other bifidobacterial species are present in only one or two of the habitats (Biavati, B. et al, (1984) Microbiologica 7, 341-345; Biavati, B. (1986) Microbiologica 9, 39-45; Crociani, F. et al, (1973) Monbl. Bakteriol. Parasitenkd. In Stammionskr. Hyg. Abt 1 Orig. Erasmus A 223, 298-302) . Also species specificity has been observed. B . magnum and B . cuniculi have been found in rabbit faeces only, while B . dentium is consistently found in dental caries of man.
  • the presence of a thick (multi-layered) cell wall generally forms a barrier for the uptake of exogenous DNA molecules.
  • the invention relates to a method for weakening the cell wall of bifidobacteria and making the bacteria permeable to nucleic acid (DNA in particular) which avoids the necessity to remove chemicals and/or enzymes which are used in conventional procedures for the introduction of DNA, prior to transformation of the bacteria.
  • bifidobacteria are important for human (and animal) health.
  • the potential usefulness of bifidobacteria for human and animal health are thought to stem from their capacity to i) contribute to the resistance against bacterial infections (Kaloud, H. & Stogmann, W. (1969) Archieves Kinderheilk. 177, 29-35) by stimulation of the immune response, ii) prevent and treat diarrhea caused by antibiotics (Mayer, J.B.
  • oligosaccharides in particular fructo-oligosaccharides has been shown to stimulate the growth in the bowl of bifidobacteria.
  • the stimulation of growth is accompanied by a reduction of the faecal pH, of ⁇ -glucuronidase and azoreductase activities, and of indole, serum cholesterol and triglyceride levels as wel as blood pressure of elderly people with hyperlipaemia (Tanaka, R. et al (1983) Bifido ⁇ bacteria Microflora 2, 17-24) .
  • Homma Homma (Homma, N.
  • Bifidobacteria are also marketed for therapeutic purposes by pharmaceutical companies e.g. for treatment of digestive disturbances in bottle-fed infants, enterocolitis, constipation, disturbed balance of the intestinal flora after treatment with antibiotics, and for the promotion of intestinal peristalsis (Rasic, J.Lj. & Kurmann, J.A.
  • Foreign DNA can, in principle, be introduced and stably maintained as part of the chromosome or extra-chromosomally, on an autonomously replicating element called episome or plasmid.
  • a gene of interest can be introduced in vivo into a bacterium by conjugation, a process which involves the mating of two bacteria and concomittant transfer of DNA from one type of bacterium (donor) to another bacterium (recipient) .
  • donor bacterium
  • recipient bacterium
  • a second method for in vivo introduction of foreign DNA is based on the use of bacteriophages which can transfer genetic information to a recipient bacterium by a process called transduct; etc.
  • Methods for gene-transfer by the recombinant DNA technique are all based on the introduction into an organism (e.g. a bacterium) of a DNA fragment carrying the genetic information for a given tra: ., which is connected to a carrier DNA molecule, called vector.
  • the vector is a circular molecule which can replicate extra-chromosomally as a multi-copy plasmid in the organism of choice.
  • protoplasted bacteria are bacteria from which the outer cell wall has been (partially) removed, in many cases by treatment with specific enzymes, sometimes in the presence of chelating agents.
  • the present invention is also related to a method of introducing DNA without the need of (partially) removing the bacterial cell wall by enzymatic and/or chemical treatment and the subsequent regeneration of protoplasted cells.
  • a break-through in the development of gene-transfer systems for LAB was realized by Chassy and Flickinger (Chassy, B.M. & Flickinger, J.L. (1987) FEMS Microbiol.
  • Electroporation involves a high-voltage electric discharge through a suspension of cells to induce transient 'pores' in the cell membrane through which DNA can enter the bacterium. Transformation of LAB strains with plasmid DNA by electroporation has been reported for Lactococcus lactis (Harlander, S. (1987) In: J.J. Ferretti & R.C. Curtiss (Eds, Streptococcal Genetics pp 229-233. Washington DC: A.S.M. Publications), L . casei (Chassy, B.M. & Flickinger, J.L.
  • the present invention provides elegant selection criteria for suitable plasmids and additionally provides very suitable plasmids for such transformations.
  • Plasmids appear to be present ubiquitously in bifido ⁇ bacteria. Plasmids were found in 70% of the strains of B . longum, the predominant species in the human intestine, in B . globosum (22% of strains) , the most common species in animals, and in 67% of the B . indicum strains, a species exclusively found in asiatic honybee hind-guts. Interestingly, strains of B . infantis, which is the species most closely related to B . longum, do not harbour plasmids, even when the two species were isolated from the same specimen.
  • a plasmid-derived vector is to be used for the intro-duction of exogenous DNA into Bifldobacterium, such a plasmid has to be able to replicate in bifidobacteria.
  • three types of plasmid may be used for this purpose.
  • Bifidobacteria and other LAB's like e.g.
  • Lactobacillus delbrueckii subsp. bulgaricus are considered as GRAS (generally Recognized &s 5_afe) organisms that can be safely used in human and animal food for the fermentation and/or preservation of food, or as food additives. If a genetically engineered microorganism is to be used for such purposes, regulatory authorities are likely to request a full description of the genetic material used for modification of the bacteria. It is also to be expected that acceptance of a modified organism will be enhanced by making use of a vector containing genes and other DNA sequences that already exist in the host bacterium, or are derived from sources that are evolutionary closely related, and/or from an other GRAS organism.
  • Bifidobacteria belong to a subclass of the Gram-positive bacteria, the genome of which is very GC-rich. Other genera of this group include Mycobacterium, Corynebacterium and Streptomyces.
  • EMBL-GenBank shows that considerable similarity exists between these plasmids. In particular, functions involved in replication are strikingly similar. These results suggest that such plasmids can replicate in related organisms, including Bifldobacterium.
  • iii) There exists a plethora of plasmids, mostly derived from Gram-positive bacteria that replicate by a so-called rolling circle replication (RCR) mechanism.
  • RCR-type plasmids are Staphylococcus aureus plasmids pC194 and pE194, and derivatives (for a review, see Gruss, S. & Ehrlich, S.D. (1989) Microbiol. Rev.
  • Plasmids replicating by the same mechanism usually are found to be incompatible if their repli ⁇ cation elements are evolutionary related (Novick, R. Microbiol. Rev. 51, 381-395) .
  • two plasmids that replicate by the same mechanism cannot co-exist in the same bacterium, unless a selection pressure is exerted to maintain the plasmids.
  • the invention comprises a method which avoids possible complications of incompatibility by using bifidobacterial strains carrying no endogenous plasmids.
  • An overnight culture of bacteria is diluted (50-fold) in MRS broth with 1% glycine or 0.2M D,L-threonine and incubated without further shaking at 37°C.
  • Cells are harvested in mid-log phase, chilled on ice, and washed twice with 5mM sodium phosphate (pH 7.4) - ImM MgCl 2 • Cells are resuspended in l/lOOth of the original volume of ice-cold electroporation buffer (0.9M sucrose, 15mM sodium-phosphate buffer (pH 7.4), and 3mM MgCl 2 , at a density of approximately 10 10 CFU/ml. Plasmid DNA (5 ⁇ g) is mixed with 50 ⁇ l of the cell suspension in an Electroporation Apparatus and subjected to an electric pulse.
  • the cell suspension is directly diluted with 450 ⁇ l of MRS broth and incubated for 1.5 h at 37°C to allow expression of the antibiotic resistance marker.
  • transformed bacteria can be directly selected on MRS agar plates containing an appropriate concentration of the antibiotic, or must first be incubated on agar plates containing sub-inhibitory concentrations (e.g. 0.5 ⁇ g/ml) of the antibiotic, after which they can be selected by replica plating at the selective (e.g. 5.0 ⁇ g/ml) concentration of the antibiotic.
  • Transformants generally are visible after 24 to 48 h of anaerobic incubation at 37°C.
  • lactis have been unsuccesful.
  • use was made of a vector derived from L . delbreuckii subsp. bulgaricus and a mutant strain deficient in restriction/modification (Sasaki, T. et al, FEMS Microbiol Rev. (1993) P8) .
  • very few strain e.g. CNRZ 32
  • have been shown to be efficiently and reproducibly transformable by electroporation see e.g. Bhowmik, T. & Steele, J. (1993) J. Gen. Microbiol. 139, 1433-1439
  • the frequency with which Ery R colonies were obtained was only about 3-6 CF ⁇ / ⁇ g pAM ⁇ l. Such a low frequency of transformation, if it had occurred, would preclude transformation of bifidobacteria with ligated DNA or with non-autonomously replicating DNA.
  • Lactobacillus helveticus and species from the Lactobacillus acidophilus groups A and B i.e. L . acidophilus, L . crispatus, L. amylovorus, L . gallinarum, L . gasseri and L . johnsonii ) .
  • Bifldobacterium species are transformed with a plasmid-derived vector which contains a selectable marker, wherein said vector is able to autonomously replicate in said organisms, but is not found in said organisms prior to transformation.
  • the vector may also contain other foreign DNA sequences which will modify or enhance expression of foreign DNA.
  • the invention provides a method of introducing nucleic acid into a microorganism, comprising inducing limited autolysis of the microorganism to improve the permeability of the cell wall of the microorganism for said nucleic acid, contacting the microorganism directly or indirectly with said nucleic acid to transform the microorganism and culturing the resulting transformants.
  • the invention provides a method for introducing at least one nucleic acid into a microorganism comprising the steps of a) treating the cell wall of the microorganism to become less impermeable to said nucleic acid, by resuspending these micro ⁇ organisms in so-called electroporation buffer, b) contacting the microorganism indirectly or directly with the nucleic acid to be introduced, and c) culturing the resulting transformants, whereby step a) is carried out by inducing limited autolysis of the microorganism.
  • the invention further provides a method as defined above, further comprising subjecting the microorganism to high voltage electric discharge.
  • the invention provides a method for introducing at least one nucleic acid into a microorganism comprising the steps of a) treating the cell wall of the microorganism to become less impermeable to said nucleic acid, by resuspending these micro ⁇ organisms in so-called electroporation buffer, b) subjecting the microorganism to a high voltage electric discharge, c) contacting the microorganism indirectly or directly with the nucleic acid to be introduced, and d) culturing the resulting transformants, whereby step a) is carried out by inducing limited autolysis of the microorganism.
  • the invention provides a method for introducing at least one nucleic acid into a lactic acid bacterium comprising the steps of a) treating the bacterial cell wall to become more permeable to said nucleic acid, b) subjecting the lactid acid bacterium to a high voltage electric discharge, c) contacting the bacterium with the nucleic acid to be introduced, and d) culturing the resulting transformants, whereby step a) is carried out by inducing limited autolysis of the lactic acid bacterium.
  • the microorganism is preferably kept in electroporation buffer to induce limited autolysis of the microorganism.
  • said electroporation buffer has low molarity and contains an osmotic stabilizer.
  • said osmotic stabilizer is selected from the group consisting of sucrose, sorbitol, mannitol, glycerol and polyethyleneglycol .
  • said buffer has a salt concentration of below lOmM, preferably of from about 0.1 to about 2mM, most preferably of from about 0.5 to about ImM.
  • said limited autolysis is carried out at a pH of from about 4 to about 8; at a temperature of from -196°C up to 37°C, preferably at about 0-37°C, more preferably at about 0-10°C, for a time period of from 10 min to 100 hours, preferably from about 1 to about 30 hours.
  • the microorganism is a recalcitrant.
  • the microorganism is a lactic acid bacterium, in particular a recr ' citrant lactic acid bacterium.
  • the bacterium .-. be transformed by the method of this invention is a Bifldobacterium species.
  • the CG content of the nucleic acid to be introduced corresponds with the CG content of the bacterium into which it is to be introduced.
  • nucleic acid to be introduced refer to all nucleic acid to be introduced and maintained in the recipient bacterium, i.e. both vector nucleic acid and insert nucleic acid.
  • at least a significant part of the nucleic acid to be introduced is evolutionary related to the microorganism into which it is to be introduced, and preferred that the nucleic acid comprises at least a part carrying the genetic information for providing the microorganism with an improved property.
  • the nucleic acid comprises at least a part encoding a protein to be expressed by the microorganism.
  • This invention also provides a transformed recalcitrant microorganism obtainable by any one of the above methods of the invention, in particular a transformed recalcitrant lactic acid bacterium, more preferably a transformed recalcitrant Bifldobacterium or Lactobacillus species .
  • the invention also embraces the use of such a transformed recalcitrant microorganism, in particular in the production or formulation of a food product, preferably a dairy product.
  • bacteria of the genus Bifldobacterium are transformed with plasmid vector DNA.
  • plasmid DNA is used that originates from Bifldobacterium, more specifically from Bifldobacterium longum.
  • a strain of Bifldobacterium animalis is used harbouring no detectable endogenous plasmids.
  • the technique of electroporation is used to introduce foreign DNA into bacteria, as this technique has proved most reliable and efficient for the introduction of DNA into other types of bacteria.
  • bacteria are used that are fragilized by limited autolytic treatment. This mild autolytic treatment not only obviates the need of irreproducible and time-consuming regeneration often seen after protoplastation, but also allows to introduce foreign DNA by means of electroporation without further treatment (removal of protoplasting enzymes like lysozyme, mutanolysin and/or chemicals such as penicillin) . In this way the process of transformation is made better reproducible and less time consuming.
  • sucrose is used as an osmotic stabilizer, but other osmotic stabilizers which are commonly used by people skilled in the art, such as e.g. sorbitol, mannitol, PEG may be used as well.
  • buffers for autolysis are used in the examples citrate buffer pH 5, citrate buffer pH 6, phosphate buffer pH 4.5, phosphate buffer pH 6, Hepes buffer pH 6 and Hepes buffer pH 7.4.
  • other compounds which buffer in the pH range from 4 to 8, such as for example cacodylate, TRIS, malate buffer can ' be used as well for the autolysis procedure.
  • the use of buffers for autolysis at concentrations with low molarity is to be preferred.
  • limited autolysis leading to electroporation-permeable bacteria is carried out at low temperatures (e.g by storing the bacteria at temperatures between -196°C and 37°C) , preferably 4°C (e.g. by placing the bacteria resuspended in ImM buffer in a refrige ⁇ rator at 4°C) for a period of 0-25 h, or longer depending on the bacterial strains.
  • This treatment causes no perceptible loss of viability or visible changes of the morphology of the bacteria.
  • Bifldobacterium bacteria are first cultivated overnight at 37°C in MRS broth (Difco; de Man, J.C. et al, (1960) J. Appl.
  • Sucrose is autoclaved separately and then added to autoclaved MRS broth.
  • bacteria are diluted, e.g. twentyfive-fold (20 ml overnight culture in 500 ml) of the same medium and incubated at 37°C for several hours. After incubation till a density at 695 nm is reached of e.g. about 0.2, bacteria are chilled on ice, collected by centrifugation at 0-4°C (15 min at 2100 x g) and washed twice with ice-cold 0.5M sucrose.
  • the pellet of bacteria is resuspensed in 1.8 ml ice- cold 0.5M sucrose.
  • Three ⁇ l of lOOmM ammonium citrate buffer, pH 6.0 are added to 300 ⁇ l of the bacterial suspension, the mixture gently swirled and placed in a refrigerator for 3 h.
  • the efficiency of transformation may vary, but can easily be optimized, e.g. by altering the duration of incubation of the bacteria in the presence of the autolysis inducing buffer. A change of the temperature at which autolysis takes place may also affect the transformation efficacy.
  • 80 ⁇ l of cell suspension obtained as described before is mixed with 2.5 ⁇ l of vector DNA (about 1.5 ⁇ g) , the mixture is gently swirled and transferred into a pre-cooled Gene Pulser disposable cuvette (inter-electrode distance 0.2 cm) .
  • a single pulse of 11000 V/cm is delivered immediately (200 ⁇ parallel resistor; 25 ⁇ F capacitance settings; Gene Pulser and Pulse Controller from Bio Rad) .
  • the cell suspension is immediately diluted in 800 ⁇ l of MRS broth supplemented with 0.05% cysteine and 0.5M sucrose.
  • bacteria are plated on selective medium agar (1.4% Daishin agar) plates (MRS + 0.05% cysteine + 0.5M sucrose, plus an appropriate concentration of antibiotic) . Transformants are visible after 2-3 days of anaerobic incubation at 37°C. It should be emphasized that, depending on the Bifldobacterium strain used, other conditions of electroporation (e.g. a higher or lower voltage) , and of growth of the bacteria following electroporation, may be needed to obtain optimal results.
  • the plasmid vector pDG7 based on the cryptic plasmid pMBl, which originates from Bifldobacterium longum B2577 (Matteuzzi, D. (1990) Letters Appl. Microbiol. 11, 220-223; Figure 1A) is used.
  • Plasmid pDG7 contains an ampicillin resistance marker for selection in E. coli and a chloramphenicol resistance marker suitable for selection in may different bacterial species such as Escherichia coll, Staphylococcus, Bacillus, Lactococcus and Lactobacillus .
  • all transformants contain a plasmid of the expected size.
  • the Bifldobacterium animalis transformants show the same carbohydrate fermentation profile as untransformed Bifldobacterium animalis, when tested in the API CH50L gallery (API System, Montalieu Verce, France) .
  • the structure of the plasmid DNA was further analyzed by restriction enzyme analysis . and shown to be identical to that of pDG7.
  • Bifldobacterium animalis, Bifldobacterium breve, Bifldobacterium bifidum, Bifldobacterium infantis and Bifldobacterium longum are used as recipient bacteria, and pDG7 as the transforming vector.
  • chloramphenicol resistant colonies are found after electroporation, under the conditions of the experiment given in the previous paragraph.
  • Transformants obtained by this method contain a plasmid having a size and structure indistinguishable from that of pDG7.
  • Bifldobacterium animalis is transformed with plasmids pECM2 and pEBM3 (Fig. IB), containing the Escherichia coli plasmid pSUP102, the resistance markers for kanamycin and chloramphenicol, and a replicon for Corynebacterium (Kalinowsky, J. pers. communication) .
  • Chloram ⁇ phenicol resistant transformants obtained after electroporation of Bifldobacterium animalis under the conditions of the experi- ment as indicated in the previous paragraph, contain a plasmid with the same size and structure as pECM2, the starting vector.
  • a vector that can integrate into the Bifldobacterium chromosome is used instead of an autonomously replicating vector like pDG7.
  • Such an integration vector will preferably contain a DNA fragment from Bifldobacterium of preferably more than 400 nucleotides, to allow homologous recombination at a predetermined site.
  • integration of the vector containing the DNA fragment of Bifldobacterium is facilitated by making use of a vector from which the gene(s) encoding the replication protein(s) but not the origin of replication are left out, and supplying the replication protein (s) encoding gene(s) in trans on a compatible plasmid.
  • the absence from the integrant of a full set of replication functions is likely to increase the genetic stability of the recombinant organism.
  • a food-grade selection marker is used instead of an antibiotic resistance marker.
  • food-grade markers may be used, e.g. genes from GRAS organisms, like Bifldobacterium, Lactococcus and Lactobacillus that can confer a dominant selectable phenotype, such as those coding for enzymes involved in (poly) saccharide metabolism (lactose, xylose, starch, inulin) or genes encoding proteins that render the bacterium resistant towards specific drugs like trimetoprim.
  • microorganisms or transformants obtainable by the methods according to the invention can basically be used in all applications of the untransformed organisms.
  • a gene encoding a useful homologous or heterologous protein is introduced into the microorganism, the microorganism can of course be used for the production of said protein.
  • the many applications of the microorganisms are of course clear to the man skilled in the art now that a method for transforming them has been disclosed in the present invention.
  • bifidobacteria are relatively resistant to low pH and to bile acids they can pass the stomach and small intestine without severe loss of viability. They thus serve as ideal vehicles for the introduction of antigens (oral immunization programs), micronutrients like iron, iodine, and vitamins (e.g. group B vitamins or vitamin A) .
  • Figure 1 is a diagrammatic representation of vectors used for transformation.
  • Figure 2 shows an analysis of plasmid DNA content of trans ⁇ formed Bifldobacterium animalis .
  • Figure 3 shows the effect of temperature and duration of autolysis on transformation efficiency of Bifldobacterium animalis .
  • the efficiency is reflected by the number of trans ⁇ formants after introduction of 1 ⁇ g pDG7 plasmid vector DNA.
  • Figure 4 shows the effect of voltage applied on trans ⁇ formation frequency of Bifidobacterium animalis .
  • Autolysing cells were mixed with pDG7 DNA in a pre-cooled 0.2 cm cuvette and subjected to an electric pulse as is indicated. Settings of the Gene Pulser and Gene Pulse controller (Bio-rad) were 25 ⁇ F and 200 ⁇ , respectively.
  • the efficiency of transformation is reflected by the number of transformants obtained per ⁇ g of vector DNA.
  • Bifldobacterium animalis was transformed by electroporation at two different voltages (6000 and 10000 V/cm) after autolysis of the bacteria in buffers of different composition.
  • Twenty ml of an overnight culture of Bifldobacterium animalis in MRS (Difco) containing 0.05% cysteine and 0.5M sucrose is diluted in 500 ml of the same medium and incubated, anaerobically, at 37°C till a density at 695 nm of 0.19 (4-5 h) .
  • the bacteria are chilled by placing them in ice-cold water, centrifuged for 15 min at 2100 x g at 4°C, and washed twice with ice-cold 0.5M sucrose.
  • the bacteria are gently resuspended in 1.8 ml 0.5M sucrose.
  • the resuspended bacteria are divided into 6 equal parts and placed in a 1.5 ml Eppendorf tube.
  • To 300 ⁇ l of resuspended bacteria is added 3 ⁇ l of the following solutions: A) lOOmM ammonium citrate buffer pH 5; B) lOOmM ammonium citrate buffer pH 6; C) lOOmM potassium phosphate buffer pH 4.5; D) lOOmM potassium phosphate buffer pH 6; E) lOOmM Hepes buffer, adjusted with NaOH to pH 6; F) lOOmM Hepes buffer adjusted with NaOH to pH 7.4.
  • the tubes are placed in a refrigerator. After 16 h, 80 ⁇ l of bacterial suspension is mixed, with gentle swirling, with 2.5 ⁇ l of pDG7 DNA.
  • the preparation of pDG7 DNA contains 1 ⁇ g of pDG7 DNA isolated from E . coli and purified by QIAGEN chromatography according to the recommendations of the supplier (DIAGEN) and 0.4 ⁇ g pDG7 DNA isolated from Bifldobacterium animalis previously transformed with pDG7.
  • the mixture is placed in a pre-cooled 0.2 cm cuvette, and subjected to an electric pulse (10000 V/cm; 200 ⁇ parallel resistor; 25 ⁇ F capacitance settings; GenePulser and PulseController from Bio-Rad) .
  • Bifldobacterium animalis was transformed with pDG7 DNA.
  • the same protocol was followed as given in EXAMPLE 1 except for a few modifications.

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EP95921168A 1994-06-17 1995-06-16 Verfahren zum einfuhren von genetisches material in mikroorganismen und damit hergestellten transformanten Withdrawn EP0765397A1 (de)

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ATE290403T1 (de) * 1997-06-30 2005-03-15 Roussy Inst Gustave Verbesserung der verabreichung der nukleinsäure in zellen der plurizellulären eukaryotischen organismen und kombination zur durchführung des verfahrens
EP1110555A1 (de) * 1999-12-22 2001-06-27 Societe Des Produits Nestle S.A. Anti-adhäsives Agens für die pathogene Hautflora
US7060687B2 (en) 2001-02-07 2006-06-13 Genmont Biotechnology Co. Live vaccines for allergy treatment
CA2442004A1 (en) 2001-03-23 2002-10-03 David J. Kyle Microbial feeds for aquaculture and agriculture
DE10119901A1 (de) 2001-04-23 2002-10-24 Amaxa Gmbh Schaltungsanordnung zur Einbringung von Nukleinsäuren und anderen biologisch aktiven Molekülen in den Kern höherer eukaryontischer Zellen mit Hilfe elektrischen Stroms
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TWI353252B (en) 2004-04-28 2011-12-01 Cms Peptides Patent Holding Company Ltd Biologically active peptide vapeehptllteaplnpk der
ES2465467T3 (es) 2004-06-14 2014-06-05 Lonza Cologne Ag Procedimiento y disposición de circuito para el tratamiento de material biológico
KR101381337B1 (ko) 2005-07-26 2014-04-11 씨엠에스 펩티드 패턴트 홀딩 컴퍼니 리미티드 신규한 생물학적 활성 펩티드 및 그의 신규한 용도
AU2007286434B2 (en) * 2006-08-18 2014-03-13 Société des Produits Nestlé S.A. Genetic remodeling in Bifidobacterium
WO2017070196A1 (en) * 2015-10-21 2017-04-27 The Methodist Hospital System Cellular vectors for delivery of therapeutics
WO2024204681A1 (ja) * 2023-03-31 2024-10-03 株式会社ヤクルト本社 ビフィドバクテリウム属細菌の形質転換方法

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JPS63216475A (ja) * 1987-03-06 1988-09-08 Meiji Milk Prod Co Ltd 乳酸桿菌属細菌の自己溶解活性誘導法
NL8700890A (nl) * 1987-04-15 1988-11-01 Tno Transformatie van lactobacillus, daarbij bruikbare recombinante plasmiden, getransformeerde lactobacillus species en gebruik daarvan.
EP0311469A3 (de) * 1987-09-02 1990-05-30 Plant Genetic Systems N.V. Transformierte Milchsäurebakterien
EP0319690A1 (de) * 1987-11-24 1989-06-14 Nisshin Flour Milling Co., Ltd. Verfahren zum Einbringen von Fremd-DNA in bestimmten Bakterien
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