RU2464317C2 - Method of chromosomal integration and replacement of dna sequence in clostridia - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves transformation of a Clostridium acetobutylicum cell by a vector containing: a replicon origin enabling its replication in C. acetobutylicum; a replacement cartridge containing a first marker gene surrounded by two sequences homologous to selected sites around the DNA target sequence, enabling recombination of said cartridge; a second marker gene representing upp counter-selection marker. The cells expressing the first marker gene are selected with the cartridge integrated in their genome. The cells not expressing the second marker gene with the eliminated said vector are selected.

EFFECT: invention enables producing the transformed Clostridium acetobutylicum cell which is genetically stable and marker-free.

31 cl, 6 dwg, 4 ex

Description

BACKGROUND OF THE INVENTION

Clostridia (Clostridia) are gram-positive, anaerobic bacteria with a low GC content, widely used in industry due to their ability to produce solvents, in particular butanol, ethanol and acetone, as well as diols like 1,3-propanediol, organic acids like acetic, butyric or lactic acid, and in vaccines.

The construction of recombinant Clostridia is an important part of the development in the art. In order to improve the possibilities of their use in industry, Clostridium strains are genetically modified.

To carry out these modifications, the most commonly used technique for all types of microorganisms is homologous recombination. Transformation and homologous recombination in certain microorganisms is comprehensively described in the art. See for example (Datsenko and Wanner; PNAS, 2000) and (Fabret et al., Molecular Microbiology, 2002).

Clostridia is inherently incapable of transformation, and current methods for their transformation are ineffective and do not allow multiple mutations to be introduced. This impedes industrial development in this area.

Typically, Clostridia produce extracellular DNases and restriction enzymes that destroy foreign DNA before and after its introduction into cells in order to carry out the transformation. The classical methods based on the introduction of PCR fragments that work well in many microorganisms, such as E. coli or yeast, are not performed in these microorganisms, because the half-life outside the cell and inside the cell of such a DNA construct that undergoes recombination is too short and recombination efficiency is generally low. In other microorganisms, these difficulties are circumvented by the use of vectors that replicate in the host, thereby increasing the likelihood of a recombination event. However, after the completion of the recombination event, the vector, now carrying the intact sequence of the target DNA, must be eliminated. This problem has been resolved in Lactococcus lactis (Biswas et al., J. Bacteriol., 1993) by using temperature-sensitive replicons that can be eliminated at non-permissive temperature. None of the vectors with these characteristics are currently available for Clostridia. Therefore, the construction of mutants in Clostridia to date has been very laborious and often unsuccessful.

Inactivation of Clostridia genes has been reported in the following articles (see table 1).

Table 1 Strain Genotype Link Clostridium acetobutylicum PJC4BK buk-, MLS ^R Green et al, 1996 Clostridium acetobutylicum PJC4PTA pta-, MLS ^R Green et al, 1996 Clostridium acetobutylicum PJC4AAD aad-, MLS ^R Green and Bennett, 1996 Clostridium perfringens SM101 and F4969 Δ cpe, CatP Sarker et al, 1999 Clostridium perfringens strain 13 Δ luxS Ohtani et al, 2002 Clostridium acetobutylicum SKO1 Δ spoA, MLS ^R Harris et al, 2002 Clostridium perfringens type A Δ spo0A Huang et al, 2004 Clostridium perfringens SM101 ccpA-, CatP Varga et al, 2004 Clostridium acetobutylicum ATCC 824 buk- Δ SpollE, buk-, CatP WO 2006/007530

To date, gene inactivation has been performed in Clostridia via transformation using circular DNA that could not replicate in target strains. Since DNA restriction enzymes and endonuclease DNAs present in Clostridia quickly destroy the introduced DNA and, as a rule, the recombination frequency in this genus is not very high, obtaining mutants was very difficult.

In addition, all the recombinant strains described so far (see above) are resistant to MLS (macrolide-lincosamide-streptogramin) or chloramphenicol, and the corresponding marker genes cannot be removed after the recombination event. This limits the number of possible recombinations by the number of available resistance markers in these bacteria to a maximum of 3. In addition, for the industrial use of these bacteria, it would be useful to have markerless strains in order to avoid the release of antibiotic resistance genes into fermentation media.

Moreover, some of these strains resulting from single recombination events have the disadvantage that they will not be stable if cultivated without any selection pressure.

Therefore, the state of the art is such that there is still a need for a method for transforming Clostridia with high efficiency, with an easy step to select recombinant strains, which allows subsequent replacement of DNA sequences in the same strain, which leads to the production of recombinant Clostridia being genetically stable and markerless.

The present invention relates to a new method for replacing or deleting a DNA sequence in Clostridia, easy to implement and applicable on an industrial scale. This method is useful for routine modification of several gene loci in Clostridia.

This method is based on the use of a replicative vector useful for transforming Clostridia with high efficiency.

Using this new method, an unlimited number of mutations can be introduced into the genome by eliminating resistance cassettes from the genome and reusing them in successful DNA sequence replacement rounds.

The effective introduction of multiple mutations in Clostridia should allow the industry to improve existing industrial strains and develop new methods.

DESCRIPTION OF THE INVENTION

The invention provides a method for replacing a target DNA sequence by homologous recombination in Clostridia, comprising:

- transformation of the specified strain by a vector containing:

- the origin of replication, allowing its replication in Clostridia, and

- substitution cassette containing the first marker gene surrounded by two sequences homologous to selected regions around the target DNA sequence, allowing the recombination of this cassette, and

- second marker gene,

- selection of strains with the indicated cassette integrated into their genome, which express the first marker gene,

- selection of strains with eliminated the specified vector, which do not express the second marker gene.

All molecular biological techniques used to implement the invention are fully described in Sambrook, Fritsch and Maniatis, "Molecular cloning: a laboratory manual", 2nd edition, Cold Spring Harbor Laboratory Press, 1989.

As used in the context of the present invention, the term “replacement” of a target DNA sequence means that a different sequence than the original one is introduced into the locus of the target DNA sequence.

According to the invention, a DNA sequence is defined as a gene or intergenic sequence. Both of them may contain promoter or regulatory sequences.

The expression “target DNA sequence” means any structure of interest selected by one of ordinary skill in the art from a gene, intergenic region, promoter or regulatory sequence. This designates, in particular, genes encoding proteins of interest, for example, enzymes involved in cellular metabolism.

The replacement / insertion DNA sequence may be coding or not. It can be a mutated sequence of a target gene, a promoter or regulatory sequence and / or a marker, such as an antibiotic resistance gene or a staining enzyme. It may be longer or shorter than the replaced sequence depending on the distance separating the two homologous regions.

Due to the presence of the insert, the expression of the target gene is usually impaired, partially or completely suppressed or enhanced. Replacing the sequence of the target DNA with a sequence close to the original, but containing mutations, leads to real expression of the mutated protein, promoter or regulatory sequence.

If, as a result of replacing the target DNA sequence, complete elimination of the indicated DNA sequence occurs, the gene is qualified as "deleted".

The expression "homologous recombination" refers to the event of replacing a DNA segment with another segment that has identical regions (homologous) or close to homologous. This event is also called DNA crossover.

The term "transformation" refers to the incorporation of an exogenous nucleic acid into a cell, and such an "acquisition" of new genes is temporary (if the vector carrying the genes is "cured") or permanent (if the exogenous DNA integrates into the chromosome).

The term "vector" refers to an extrachromosomal element carrying genes or cassettes, which usually exists in the form of circular double-stranded DNA molecules, but can also be a single-stranded DNA molecule. Both terms "vector" and "plasmid" are used independently.

The vector of the invention is a replicative vector. It contains at least one origin of replication and preferably several replication initiation sites, which allows it to be functionally active in different forms.

A particularly preferred vector may contain two replication initiation sites:

- Ori, functionally active in E. coli,

- RepL from pIM13 originating from B. subtilis, functionally active in Clostridia (Mermelstein et al., Biotechnology, 1992).

The expression “sequences homologous to the target DNA sequences” refers to sequences having greater sequence similarity to selected portions of the target sequence.

The term "marker gene" refers to a sequence encoding a marker protein under the control of regulatory elements functionally active in Clostridia. Such proteins are well known in the art. For example, one skilled in the art may use an antibiotic resistance gene, a fluorescent or staining marker, or an auxotrophicity marker. Examples of useful marker genes will be given below.

Now, after the implementation of the recombination event, the vector carries the intact sequence of the target DNA and therefore must be eliminated. Elimination of the replicative vector usually occurs in subsequent cultures of clones as a result of negative or positive selection of clones from which this vector is eliminated. The elimination of the vector can also be an active step in this method using endonucleases that specifically cleave the DNA sequences present in the vector. Once the vector is "cured", the strains no longer express the second marker gene and can be selected by this characteristic.

In a specific embodiment of the invention, the second marker gene is an antibiotic resistance gene. Among the useful antibiotic resistance genes, one skilled in the art will know which is most appropriate. For example, you can use the following genes: the CatP gene, which gives resistance to chloramphenicol and thiamphenicol, or the MLS ^R gene, which gives resistance to erythromycin.

In a preferred embodiment of the invention, the second marker gene is a counter-selective marker gene.

A counter-selectable marker is a gene whose presence is lethal to the host microorganism in some cases, such as the presence of a related substrate. Anti-selectable markers can be used as positive selection for plasmid loss.

Preferably, the counter-selectable marker gene is a gene that restores the activity of an absent or deleted non-essential endogenous gene.

The most commonly used counter-selectable markers are genes that confer sensitivity to sucrose, streptomycin or fusaric acid. They are used to construct mutants or vaccine strains in several bacterial strains. See Reyrat et at., 1998, Infection and Immunity for a detailed discussion. Anti-selectable markers that can be used in Clostridia include genes that are sensitive to 5-fluoro-uracil (5-FU), gamma-glutamyl hydrazide (GBS), or 8-aza-2,6-diaminopurine (8ADP).

In a preferred embodiment, the counter-selectable marker is the upp gene encoding uracil phosphoribosyl transferase, which promotes the conversion of 5-fluorouracil (5-FU) into a toxic product. Cells with Upp activity cannot grow on 6-FU-containing medium.

The use of this counter-selectable marker is particularly useful when transformed Clostridia are Δupp and therefore are able to grow on a 5-FU containing medium before transformation and after elimination of the vector. Eliminated vector strains can be positively selected.

Suppressor mutations that can appear in the upp gene in the presence of 5-FU can sometimes lead to erroneous assumptions regarding plasmid loss. In a preferred embodiment of the invention, the vector further comprises a third marker, preferably an antibiotic resistance gene, which enables a second selection of antibiotic sensitive strains. Such negative selection can be used in addition to positive selection based on the upp gene.

In a preferred embodiment of the invention, the vector is eliminated by digestion with endonucleases after the implementation of the recombination event. Preferably, the vector carries DNA sequences that are recognized by restriction endonucleases and which, however, are missing from the genome of the used species of Clostridium. Therefore, the vector is specifically destroyed without loss of integrity of the Clostridium genome.

Restriction endonucleases are enzymes that cleave DNA molecules at the position of specific base sequences, the restriction site of the endonuclease. The person skilled in the art is able to determine which restriction endonuclease site is missing in the genome of the Clostridium strain of interest. Possible restriction endonucleases that can be used for C. acetobutylicum are AscI, FseI, NotI, SfiI, SrfI. In another embodiment, meganucleases that recognize large (12-45 bp) target DNA sites, such as I-SceI, HO or I-CreI, can be used.

In a preferred embodiment of the invention, the Clostridium strain to be transformed carries at least one gene encoding an endonuclease in its genome that recognizes the restriction site of the endonuclease present in the vector. It is possible that the expression of restriction endonuclease is under the control of an inducible promoter.

An inducible promoter is a DNA element that allows expression of a target sequence due to the addition of an appropriate inducer. For example, the inducible promoter system in Clostridium, known to the person skilled in the art, is described in Girbal et al., 2003, Appl. Env. Microbiol. 69: 4985-8.

After the recombination event has occurred and before screening for the strains in which the vector is to be eliminated, expression of a restriction endonuclease can be induced. Restriction endonuclease will cleave the vector present in Clostridia, which will lead to its elimination.

It is possible that a gene encoding a restriction endonuclease can be inserted into the genome before introducing a vector into this strain.

In another embodiment of the invention, a restriction endonuclease encoding gene can also increase the recombination frequency before eliminating the plasmid by increasing the amount of linear DNA in the cells, which is known to undergo recombination better than ring DNA.

In a specific embodiment of the invention, the first marker gene is an antibiotic resistance gene inserted in the middle of the replacement cassette.

In a specific embodiment of the invention, this first marker gene can be deleted from the genome of transformed Clostridium strains. In particular, the first marker gene can be surrounded by two recombinase target sites and then eliminated by the action of recombinase after the act of homologous recombination.

In a preferred embodiment of the invention, the recombinase is expressed by a second vector carrying the corresponding gene, wherein said vector is introduced into Clostridia as a result of transformation.

Preferably, the recombinase target sites are FRT (Flippase Recognition Target) sequences (recognized by the flippase of the target sequence). Recombinase FLP (flippase) from Saccharomyces cerevisiae is active for a particular 34-base DNA sequence designated as FRT sequence (target for FLP recombinase). If two of these FRT sites are present, then the FLP enzyme produces double-stranded breaks in the DNA chains, exchanges the ends of the first FRT with the ends of the second target sequence, and then reconnects the changed chains. This process leads to the deletion of the DNA that lies between these two sites.

In a particular embodiment of the invention, sequences homologous to selected regions near the target DNA sequence may contain mutations affecting up to 10% base pairs, including the DNA fragment used for the recombination event.

In a preferred embodiment of the invention, the Clostridium strains to be transformed have deletions in the genes encoding restriction endonucleases. These strains are advantageous in that they are easily transformed without any prior methylation of the plasmid in vivo.

In another preferred embodiment of the invention, the Clostridium strains to be transformed have deletions in genes encoding extracellular DNAse.

In another embodiment of the invention, the Clostridium strains to be transformed have deletions in the upp gene.

In another preferred embodiment, strains of Clostridium, to be transformed are chosen among Clostridium acetobutylicum, Clostridium bejeirinckii, Clostridium saccharoperbutylacetonicum, Clostridium butylicum, Clostridium butyricum, Clostridium perfringens, Clostridium tetani, Clostridium sporogenes, Clostridium thermocellum, Clostridium saccharolyticum (now the Thermoanaerobacter saccharolyticum), Clostridium thermosulfurogenes (now Thermoanaerobacter thermosulfurigenes), Clostridium thermohydrosulfuricum (now Thermoanaerobacter ethanolicus).

A preferred strain of Clostridium is Clostridium acetobutylicum.

In a preferred embodiment of the invention, the strain of Clostridium acetobutylicum to be transformed is strain ΔCac15 having a deletion in the gene encoding the restriction endonuclease Cac 824I. This strain is advantageous in that it is easy to transform without any prior methylation of the plasmid in vivo.

In another preferred embodiment of the invention, the strain of Clostridium acetobutylicum to be transformed is a strain having a deletion in the upp gene designated Δ upp. As a result, transformation of this strain with a vector carrying the upp gene allows selection of strains that are sensitive to 5-containing medium, and then positive selection of strains that have lost the plasmid, which are more insensitive to S-FU-containing medium.

The strain Clostridium acetobutylicum, which must be transformed, can also be ΔCac15 Δ upp.

This method is preferably used for subsequent replacement of two or more target genes by homologous recombination in the same strain of Clostridium.

The present invention also relates to a recombinant strain of Clostridium, allowing its production in accordance with the method according to the invention. Preferably, the method of the invention can be used to produce recombinant Clostridium strains, where the cac15 gene was deleted (strain Δcac15), where the upp gene was deleted (strain Δupp) and where both the cac15 and upp genes were deleted (strain ΔCac15 Δupp). The present invention also relates to a vector for transformation of a given strain, as for example described above.

STATEMENT OF GRAPHIC MATERIALS

Figure 1. PUC18-FRT-MLS2 vector map.

Figure 2. PCons2-1 vector map.

Figure 3. PCIP2-1 vector map.

Figure 4. PCons :: UPP. Vector Map

Figure 5. PCLF1 vector map.

6. Schematic representation of the deletion method for Clostridia.

The following steps are performed sequentially:

A — amplification of two selected regions near the target DNA sequence; 1, 2, 3, 4 are PCR primers;

B - cloning of the obtained PCR fragments into the cloning vector rTORO;

C is the insertion of a marker into the StuI restriction site present in PCR primers;

D — cloning of the replacement cassette into the pCONS 2.1 BamHI site: construction of the pREP cloY vector;

E - transformation of Clostridium by the vector pREP cloY;

F - chromosome integration of the replacement cassette through double crossing over in the process of subculture;

G - screening of clones with phenotypes Ery ^R and Thiam ^S ;

H - PCR analysis for the purpose of monitoring for gene replacement and plasmid loss;

or

D '- cloning of the replacement cassette into the BamHI site pCONS :: UPP: construction of the vector pREP cloY: UPP;

E 'is the transformation of Clostridium Δupp by the vector pREP cloY: UPP;

F '- chromosome integration of the replacement cassette through double crossing over in the process of subculture;

G 'screening of clones with phenotypes Ery ^R and 5-FU ^R (Thiam ^S );

H '- PCR analysis to control for gene replacement and plasmid loss.

EXAMPLES

Example 1. Construction of vectors

1.1. Construction of pUC18-FRT-MLS2

This plasmid contains the MLS ^r gene, functionally active in Clostridia and flanked by two FRT sites and two StuI sites and useful for constructing replacement cassettes. Reverse polymerase chain reaction (PCR) was performed using Pwo DNA polymerase with pKD4 as the template plasmid (Datsenko and Wanner, 2000) and oligonucleotides PKD4.1 and PKD4.2 as primers for amplification of the plasmid region with FRT sites, but without marker Km ^r . This end-dephosphorylated fragment was later ligated to the MLS ^r gene obtained after digestion with the HindIII plasmid pETSPO (Harris et al., 2002, J. Bacteriol.) And treatment with the Klenov fragment. Then, the corresponding plasmid (pKD4-Ery1) was used as a template for amplification of the MLS ^r gene, flanked by two FRT sites and two StuI sites, in the PCR reaction using oligonucleotides FRT-MLSR-F and FRT-MLSR-R as primers and Pwo DNA polymerase. This fragment was cloned directly into pUC18 digested with SmaI (plasmid) to obtain plasmid pUC18-FRT-MLS2 (Figure 1).

PCR primers:

PKD4.1 (SEQ ID No. 1): 5'-ctggcgccctgagtgcttgcggcagcgtgagggg-3 ' PKD4.2 (SEQ ID No. 2): 5'-agcccggggatctcatgctggagttcttcgccc-3 ' FRT-MLSR-F (SEQ ID No. 3): 5'-tacaggccttgagcgattgtgtaggctggagc-3 ' FRT-MLSR-R (SEQ ID No. 4): 5'-aacaggcctgggatgtaacgcactgagaagccc-3 '

1.2. Design pCons 2.1

This plasmid contains the origin of pIM13 replication, functionally active in Clostridia (“rolling ring” replication), the catP gene giving resistance to thiamphenicol, and a unique BamHI site for cloning a replacement cassette. This plasmid was constructed using a two-step procedure from the pETSPO plasmid (Harris et al., 2002, J. Bacteriol.) To remove part of the polylinker and, among others, the BamHI and EcoRI sites. OPCR was performed using Pwo DNA polymerase with pETSPO as a template plasmid and with oligonucleotides PCONSAccI and PCONSEcoRI as primers, and the PCR product was phosphorylated and ligated. After transformation of E. coli, plasmid pCons0 was obtained. Then this plasmid was digested using BamHI to remove the spo0A cassette, the desired DNA fragment was purified and ligated to obtain plasmid pCons2-1. The pCons2-1 map is shown in FIG. 2.

PCR primers:

PCONSAccI (SEQ ID No. 5): 5'-ccggggtaccgtcgacctgcagcc-3 ' PCONSEcoRI (SEQ ID No. 6): 5'-gaattccgcgagctcggtacccggc-3 '

1.3. Construction of the pCIP2-1 Vector

In this design, the origin of replication p1M13 from pCons2-1 was replaced by the origin of replication of plasmid pSOL1, plasmid with θ-replication mechanism. For this, the origin of replication pSOL1 was subjected to PCR amplification using Pwo DNA polymerase using total DNA of C. acetobutylicum as a template and ori-3-D and ori-4-R oligonucleotides as primers. This PCR product was cloned into pCR-Bluntll-TOPO, the resulting plasmid was digested using EcoRI, and a 2.2 kb fragment was purified. Similarly, the plasmid pCons2.1 was digested using EcoRI, a 2.4 kb fragment was purified. and ligated it with 2.2 kb EcoRI fragment containing the origin of replication from pSOL1, obtaining the plasmid pCIP2-1 (Figure 3).

PCR primers:

ORI-3-D (SEQ ID No. 7): 5'-ccatcgatgggggtcatgcatcaatactatcccc-3 ' ORI-4-R (SEQ ID No. 8): 5'-gcttccctgttttaatacctttcgg-3 '

1.4. PCons :: upp vector construction

The upp gene, which has its own ribosome binding site (rbs; ribosome binding site), was cloned into pCons2.1 at the BcgI site located immediately upstream of the CatP gene in order to construct an artificial operon with upp expressed under the control of the CatP promoter. Thus, no homologous regions were introduced that would allow chromosome integration of the upp gene into the Δcac15Δupp strain in subsequent deletion experiments, in which the upp gene is used as an anti-selectable marker for plasmid loss.

The upp gene, along with its rbs, was subjected to PCR amplification (Pfu) amplification from C. acetobutylicum genomic DNA using REP-UPP F and REP-UPP R oligonucleotides as primers. 664 bp PCR product digested using PvuII, cloned into pCons2.1 digested with Bcgl, and treated with T4 DNA polymerase to obtain dephosphorylated ends. Thus, the replicative vector pCons :: UPP was obtained (see Figure 4).

PCR primers:

REP-UPP F (SEQ ID NO: 9): 5'-aaaacagctgggaggaatgaaataatgagtaaagttacac-3 '

REP-UPP R (SEQ ID No. 10); 5'-aaaacagctgttattttgtaccgaataatctatctccccagc-3 '

1.5. Construction of the pCLF1 Vector

The S. cerevisiae FLP1 gene encoding the FLP recombinase was cloned into the pCons2.1 vector under the control of a promoter and RBS from the thiolase (thI) gene from C. acetobutylicum, which causes a high level of expression in this microorganism.

The FLP1 gene was subjected to PCR amplification (Pfu) amplification using oligonucleotides FLP1-D and FLP1-R as primers and plasmid pCP20 (Datsenko and Wanner, 2000) as a template.

PCR primers:

-FLP1-D (SEQ ID No. 11): 5'-aaaaggatccaaaaggagggattaaaatgccacaatttggtatattatgtaaaacaccacct-3 '

-FLP1-R (SEQ ID No. 12): 5'-aaatggcgccgcgtacttatatgcgtctatttatgtaggatgaaaggta-3 '

FLP1-D has an extension segment at the 5'-end, including the BamIII site and the RBS sequence from thI.

Using FLP1-R, the SfoI site was introduced at the 3'-end of the PCR product.

The PCR product was digested using BamHI and SfoI and directly cloned into the pSOS95 expression vector, which was digested with the same enzymes, to obtain the plasmid pEX-FLP1 (6281 bp).

The SalI fragment (1585 bp) of pEX-FLP1 containing the FLPI-expressing cassette was cloned at the pCONS2.1 SalI site to obtain plasmid pCLF1 (4878 bp) (Figure 5).

Example 2. The deletion of the gene cas1502, encoding the restriction enzyme cas824I in Clostridium acetobutylicum

For a schematic representation of this method, see FIG. 6.

Two DNA fragments surrounding the gene encoding Cac824I (CAC1502) were PCR amplified using Pwo DNA polymerase using total DNA from C. acetobutylicum as a template and two specific pairs of oligonucleotides as primers (see Table 2). Using pairs of primers CAC 1B-CAC 2 and CAC 3-CAC 4B, DNA fragments of 1493 bp were obtained. and 999 bp, respectively. Using both primers CAC 1B and CAC 4B, the SamHI site is introduced, while primers CAC 2 and CAC 3 have complementary 5 ′ end-sequences that introduce the StuI site. The CAC 1 B-CAC 2 and CAC 3-CAC 4 B DNA fragments were combined in a PCR fusion experiment using the CAC 1B and CAC 4B primers, and the resulting fragment was cloned into the pCR4-TOPO-Blunt vector to obtain rTORO: cac15. According to the unique StuI site of rTORO: cac15, a StuI fragment of 1372 bp was introduced. from pUC18-FRT-MLS2, which carries the antibiotic resistance gene MLS ^r with FRT sequences on both sides. The cac1502 substitution cassette obtained after digestion of the obtained plasmid using BamHI was cloned at the BamHI site in pCons2-1 to obtain plasmid pREPCAC15 and to pCIP2.1 to obtain pCIPCAC15.

Plasmids pREPCAC15 and pCIPCAC15 were methylated in vivo and used to transform C.acetobutylicum by electroporation. After selection on Petri dishes for clones resistant to erythromycin (40 μg / ml), one colony from each of the transformants was cultured for 24 hours in a liquid synthetic medium with erythromycin at a concentration of 40 μg / ml and then subcultured in 2YTG liquid medium without antibiotic. Appropriate dilutions were applied to enriched clostridia agar (RCA) with erythromycin at a concentration of 40 μg / ml. For the selection of integrants that have lost the pREPCAC15 or pCIPCAC15 vectors, erythromycin-resistant clones were applied by replica both on RCA with erythromycin at a concentration of 40 μg / ml, and on RCA with thiamphenicol at a concentration of 50 μg / ml. While several colonies with the desired phenotype were obtained using pREPCAC15 transformants, no such colonies were obtained using pCIPCAC15 transformants. This fact demonstrates that the θ replication mechanism of pCIPCAC15 is less suitable for promoting double crossing over in C. acetobutylicum than the rolling ring mechanism. The genotype of erythromycin-resistant and thiamphenicol-sensitive clones was verified by PCR analysis (using CAC 0 and CAC 5 primers located outside the CAC15 substitution cassette, and CAC D and CAC R primers located inside cAC1502). Strains Δcac15 :: mls ^R that lost pREPCAC15 were isolated.

The strain Δcac15 :: mls ^{R was} transformed with the pCLF1 vector expressing the FLP1 gene encoding Flp recombinase from S. cerevisiae. After transformation and selection for resistance to thiamphenicol (50 μg / ml) in Petri dishes, one colony was cultured in a synthetic liquid medium with thiamphenicol at a concentration of 50 μg / ml and the corresponding dilutions were applied to RCA with thiamphenicol at a concentration of 50 μg / ml. Thiamphenicol-resistant clones were applied by the replica method both on RCA with erythromycin at a concentration of 40 μg / ml, and on RCA with thiamphenicol at a concentration of 50 μg / ml. The genotype of clones with sensitivity to erythromycin and resistance to thiamphenicol was checked by PCR analysis using CAS 0 and CAS 5B primers.

Two consecutive 24-hour cultivations of the strain Δcac15 were carried out with sensitivity to erythromycin and resistance to thiamphenicol to remove pCLF1. Strain Δcac15, which lost pCLF1, was isolated in accordance with its sensitivity to both erythromycin and thiamphenicol. The strain was designated as MGCΔcac15 C.acetobutylicum.

Example 3. Deletion of the upp gene encoding uracil-phosphoribosyltransferase in Clostridium acetobutylicum Δcac15

Two upstream and downstream DNA fragments from upp (CAC2879) were PCR amplified using Pwo DNA polymerase using total DNA from C. acetobutylicum as template and two specific oligonucleotide pairs as primers (see Table 3). Using pairs of primers UPP 1-UPP 2 and UPP 3-UPP 4, DNA fragments of 1103 bp were obtained. and 1105 bp, respectively. Using both the primers UPP 1 and UPP 4, the BamHI site is introduced, while the primers UPP 2 and UPP 3 have elongated sequences from the 5'-end with which the StuI site is introduced. The UPP 1-UPP 2 and UPP 3-UPP 4 DNA fragments were combined in a PCR fusion experiment using UPP 1 and UPP 4 primers, and the resulting fragment was cloned into pCR4-TOPO-Blunt to obtain pTORO: upp. According to the unique StuI site of rTORO: upp, a StuI fragment of 1372 bp was introduced. from pUC18-FRT-MLS2, which carries the antibiotic resistance gene MLS ^r with FRT sequences on both sides. The upp substitution cassette obtained by digesting the obtained plasmid using SamHI was cloned into pCons2-1 at the BamH1 site to obtain the plasmid pREPUPP.

Plasmid pREPUPP was used to transform C.acetobutylicuin MGCΔcac15 strain by electroporation without prior in vivo methylation. After selection on Petri dishes for clones resistant to erythromycin (40 μg / ml), one colony was cultured for 24 hours in a liquid synthetic medium with erythromycin at a concentration of 40 μg / ml and then subcultured in 2YTG liquid medium without antibiotic. Appropriate dilutions were applied to RCA with erythromycin at a concentration of 40 μg / ml. For the selection of integrants that have lost the pREPUPP vector, erythromycin-resistant clones were applied by replica both on RCA with erythromycin at a concentration of 40 μg / ml, and on RCA with thiamphenicol at a concentration of 50 μg / ml. The genotype of erythromycin-resistant and thiamphenicol-sensitive clones was verified by PCR analysis (using UPP 0 and UPP 5 primers located outside the replacement UPP cassette, and UPP D and UPP R primers located inside UPP). The strain Δcac15Δupp :: mls ^R , which lost pREPUPP, was isolated. The previous deletion of cac1502 was confirmed as described previously in the first example. The strain Δcac15Δupp :: mls ^R is resistant to 5-FU at a concentration of 400 μm compared with 50 μM for strain Δcac15.

The strain Δcac15Δupp :: mls ^{R was} transformed with the pCLF1 vector expressing the FLP1 gene encoding Flp recombinase from S. cerevisiae. After transformation and selection for resistance to thiamphenicol (50 μg / ml) in Petri dishes, one colony was cultured in a synthetic liquid medium with thiamphenicol at a concentration of 50 μg / ml and the corresponding dilutions were applied to RCA with thiamphenicol at a concentration of 50 μg / ml. Thiamphenicol-resistant clones were applied by the replica method both on RCA with erythromycin at a concentration of 40 μg / ml, and on RCA with thiamphenicol at a concentration of 50 μg / ml. Clone genotype with erythromycin sensitivity and resistance to thiamphenicol was tested by PCR analysis using UPP 0 and UPP 5 primers. Two consecutive 24-hour cultivations of Δcac15Δupp strain with erythromycin sensitivity and thiamphenicol resistance were performed to remove pCLF1. The strain Δcac15Δupp, which lost pCLF1, was isolated in accordance with its sensitivity to both erythromycin and thiamphenicol.

Example 4. The deletion of gene sas3535 using upp and 5-fluorouracil as a positive selection for the loss of plasmids

Two DNA fragments located upstream and downstream from the cac3535 gene encoding the second restriction-modification system of C.acetobutylicum were subjected to PCR amplification using Pwo DNA polymerase using total DNA from C.acetobutylicum as matrices and two specific pairs of oligonucleotides (see Table 4). Using pairs of primers RM 1-RM 2 and RM 3-RM 4, DNA fragments of 1 kb were obtained. and 0.9 kbp, respectively. Using both primers RM 1 and RM 4, a BamHI site is introduced, while primers RM 2 and RM 3 have complementary 5 ′ endpoint sequences by which a StuI site is introduced. DNA fragments RM 1-RM 2 and RM 3-RM 4 were combined in a PCR fusion experiment using primers RM 1 and RM 4, and the resulting fragment was cloned into the pCR4-TOPO-Blunt vector to obtain plasmid pTOPO: cac3535. According to the unique StuI rTORO: сас3535, a 1372 bp Sful fragment was introduced. from pUC18-FRT-MLS2, which carries the antibiotic resistance gene MLS ^r with FRT sequences on both sides. The CAC3535 substitution cassette obtained after digestion of the obtained plasmid using BamHI was cloned into pCons :: upp at the BamHI site to obtain plasmid pREPCAC3535 :: upp.

Plasmid pREPCAC3535 :: upp was used to transform C.acetobutylicum MGCΔcac15Δupp strain by electroporation. After selection on Petri dishes for clones resistant to erythromycin (40 μg / ml), one colony was cultured for 24 hours in a liquid synthetic medium with erythromycin at a concentration of 40 μg / ml and 100 μl of undiluted culture was applied to RCA with erythromycin at a concentration 40 μg / ml and 5-FU at a concentration of 400 μM. Colonies resistant to both erythromycin and 5-FU were applied by the replica method both on RCA with erythromycin at a concentration of 40 μg / ml and on RCA with erythromycin at a concentration of 50 μg / ml in order to verify that resistance to 5 -FU is associated with sensitivity to thiamphenicol. The genotype of erythromycin-resistant and thiamphenicol-sensitive clones was verified by PCR analysis (using primers RM 0 and RM 5 located outside the CAC3535 substitution cassette, and primers RM D and RM R located inside the cAC3535 gene). Thus, the strain Δcac15ΔuppΔcac35 :: m ^R , which lost pREPCAC3535 :: upp, was isolated.

Claims (31)

1. A method of replacing a target DNA sequence by homologous recombination in Clostridium acetobutylicum, comprising:
- transformation of a Clostridium acetobutylicum cell with a vector containing:
- the origin of replication, allowing its replication in C. acetobutylicum, and
- a replacement cassette containing the first marker gene surrounded by two sequences homologous to selected regions near the target DNA sequence, allowing the recombination of this cassette, and
- second marker gene,
- selection of cells with an integrated cassette integrated into their genome that express the first marker gene,
- selection of cells with eliminated the specified vector, which do not express the second marker gene,
where the second marker gene is the upp gene, an anti-selectable marker. 2. The method according to claim 1, where the counter-selectable marker is a gene that restores the activity of a non-essential missing or delegated gene. 3. The method according to claim 1, where the vector contains a third marker, which allows negative selection of cells with eliminated the specified vector. 4. The method according to claim 1, where the vector is eliminated as a result of digestion by endonucleases. 5. The method according to claim 4, where the vector carries DNA sequences that are recognized by restriction endonucleases and which, however, are absent in the genome of C. acetobutylicum cells used. 6. The method according to claim 4 or 5, where the used C. acetobutylicum cells carry in their genome at least one endonuclease specific for the restriction sites present in the vector, possibly expressed under the control of an inducible promoter. 7. The method according to claim 1, where the first marker gene is an antibiotic resistance gene. 8. The method according to claim 1, where the first marker gene is surrounded by two recombinase target sites. 9. The method of claim 8, where after the act of homologous recombination has occurred and the replacement cassette is integrated, the first marker gene located in this cassette is eliminated by recombinase. 10. The method according to any one of claims 8 or 9, wherein said recombinase is expressed by a gene delivered by a second vector introduced into these cells. 11. The method according to any one of claims 8 or 9, wherein said recombinase target sites are FRT (Flippase Recognition Target) sequences (recognized by the flippase of the target sequence), and said recombinase is a FLP (flippase) recombinase. 12. The method according to claim 1, where these sequences homologous to regions near the sequence of the target DNA contain mutations, including up to 10% of base pairs used for the recombination event. 13. The method according to claim 1, where C. acetobutylicum, which must be transformed, have deletions of genes encoding restriction endonucleases. 14. The method according to claim 1, where C. acetobutylicum, which must be transformed, have deletions of genes encoding DNase. 15. The method according to claim 1, where C. acetobutylicum, which must be transformed, have deletions of the upp gene. 16. The method according to claim 1, where the cells of Clostridium acetobutylicum, which must be transformed, are Δ sas15. 17. The method according to claim 1, where the cells of Clostridium acetobutylicum, which must be transformed, are Δ sas15 Δ upp. 18. A method for replacing two or more target genes by homologous recombination in the same C. acetobutylicum cell, said method according to any one of claims 1-17, being carried out sequentially two or more times. 19. A transformed Clostridium acetobutylicum cell, obtained in accordance with the method according to any one of claims 1 to 18. 20. The transformed C. acetobutylicum cell according to claim 19, where the cac15 gene was deleted. 21. The transformed C. acetobutylicum cell according to claim 19, where the upp gene was deleted. 22. The transformed C. acetobutylicum cell according to claim 19, where both the cac15 and upp genes were deleted. 23. A transforming vector containing:
- the origin of replication, allowing its replication in C. acetobutylicum, and
- a replacement cassette containing the first marker gene surrounded by two sequences homologous to selected regions near the target DNA sequence, allowing the recombination of this cassette, and
- upp gene is the second counter-selectable marker gene. 24. The vector according to item 23, where the counter-selectable marker is a gene that restores the activity of a non-essential missing or deleted gene. 25. The vector according to item 23, containing the third marker, which allows negative selection of cells with eliminated the specified vector. 26. The vector according to item 23, which is eliminated as a result of digestion by endonucleases. 27. The vector according to p. 26, carrying DNA sequences that are recognized by restriction endonucleases and which at the same time are absent in the genome of C. acetobutylicum cells used. 28. The vector according to item 23, where the first marker gene is an antibiotic resistance gene. 29. The vector according to item 23, where the first marker gene is surrounded by two recombinase target sites. 30. The vector according to clause 29, where these recombinase target sites are FRT (Flippase Recognition Target) sequences (recognized by the flippase of the target sequence), and the specified recombinase is a recombinase FLP (flippase). 31. The vector according to claim 23, wherein said sequences homologous to regions near the target DNA sequence contain mutations involving up to 10% of base pairs used for the recombination event.

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