JP2010017131A - Bacteria of genus moorella and primer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide bacteria of the genus Moorella which are available for wide uses as a host cell, and a primer which is available upon creation of bacteria of the genus Moorella which are available for wide uses as a host cell. <P>SOLUTION: Disclosed are bacteria of the genus Moorella having features that genes on chromosomes are deleted or destroyed by homologous recombination to exhibit auxotrophy, and a specific primer which amplifies a region adjacent to a gene encoding orotate phosphoribosyltransferase. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a recombinant of a bacterium belonging to the genus Morella, and in particular, a primer that can be used to produce a bacterium that belongs to the genus auxotrophy and an auxotrophic strain imparted with auxotrophy by deleting or destroying a gene on a chromosome by homologous recombination About.

  Research on new energy is being promoted to combat global warming and reduce consumption of fossil fuels. In particular, in recent years, demand for fuel has been increasing, and ethanol has a high value as a useful product.

  Using (thermophilic) acetic acid-producing bacteria such as Clostridium as raw materials from gaseous carbon dioxide, carbon monoxide, hydrogen, etc. contained in the gas discharged from gasification and reforming of industrial waste gas and woody biomass There is a technique for producing and recovering useful substances such as acetic acid and ethanol (for example, Patent Document 1).

  This technology does not use crops that can be food such as sugarcane and corn as raw materials, so there is no impact on food prices, and carbon dioxide is used as a raw material, so it can also be expected to reduce greenhouse gases. It is.

Moorella sp. HUC22-1 (Clostridial sp. No. 22-1 strain (FERM P-18852)) is a thermophilic acetic acid-producing bacterium strain that can be used in the technique of Patent Document 1, and is 45 to 65 ° C., pH 4 It can grow at 5 to 7.5, shows the highest growth at 55 to 60 ° C. and pH 5.7 to 6.7, has high ethanol productivity, and has a good balance of resistance to H ₂ , ethanol and acetic acid ( Non-patent document 1). In Non-Patent Document 2, when repeated batch culture was performed under the condition of constant control of pH 5.8 using H ₂ —CO ₂ as a substrate, the total ethanol production was 15.4 mmol / (L-reactor). It is described.
JP 2003-339371 A Sakai et al. “Ethanol production from H2 and CO2 by a newly isolated thermophilic bacterium, Moorella sp. HUC22-1” Biotechnology Letters, 2004, 26, 1607-1612 Sakai et al. “Acetate and ethanol production from H2 and CO2 by Moorella sp. Using a repeated batchculture.” Journal of Bioscience And Bioengineering, 2005, 99, 252-258

The use of thermophilic acetic acid producing bacteria for the fermentation of ethanol is compared to the case of using yeast or mesophilic acetic acid producing bacteria, because (1) the culture temperature is higher (50 to 80 ° C.). Recovery is easy, (2) The heating cost in the case of thermophilic bacteria is lower than the cooling cost in the case of yeast, etc. (3) Since the culture temperature is high, it depends on natural conditions (4) Since the culture is carried out in a high temperature and oxygen-free state, there is an advantage that there is very little risk of contamination, but it is still in the research stage and commercial. In order to realize production, it is necessary to improve the production efficiency of microorganisms.

  In order to obtain a strain excellent in ethanol production ability, the present inventors have used Mooreella sp. HUC22-1 was subjected to mutation treatment with a chemical substance such as NTG, but no strain that maintained high ethanol production after passage was obtained.

Therefore, the present inventors decided to try to produce a high ethanol production strain by molecular biological breeding that can introduce or destroy a target gene at a pinpoint. However, E.I. Unlike general bacteria such as E. coli, there have been few reports on gene transfer methods using thermophilic anaerobic bacteria as hosts. Especially, gene transfer methods using bacteria of the genus Morella as a host have never been reported. Not.

  First, we attempted to introduce a gene using a plasmid vector, which is one of the molecular biological breeding methods, but this method did not yield a strain that was confirmed to be transformed.

  Thus, the present inventors have found a homologous site and attempted recombination by a homologous recombination method, and succeeded in producing a Morella bacterium that can be used for a wide range of uses as a host cell.

  That is, an object of the present invention is to provide a bacterium belonging to the genus Morella that can be used for a wide range of uses as a host cell.

  Another object of the present invention is to provide a primer that can be used when producing a bacterium belonging to the genus Morella that can be used for a wide range of uses as a host cell.

  Furthermore, the other subject of this invention becomes clear by the following description.

  The above problems are solved by the following invention.

(Claim 1)
A bacterium belonging to the genus Morella, wherein a gene on a chromosome is deleted or destroyed by homologous recombination and exhibits auxotrophy.

(Claim 2)
The bacterium belonging to the genus Morella according to claim 1, wherein the gene on the chromosome is a gene encoding an enzyme involved in a biosynthetic pathway of a pyrimidine base, and the auxotrophy is uracil auxotrophy.

(Claim 3)
The bacterium belonging to the genus Morella according to claim 1 or 2, wherein the gene on the chromosome is a gene encoding orotate phosphoribosyltransferase.

(Claim 4)
Moorella sp. The bacterium belonging to the genus Morella according to any one of claims 1 to 3, which has been deposited at the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation, 22-1-U (reception number NITE AP-606).

(Claim 5)
A primer used in the production of an auxotrophic strain in which a gene on a chromosome has been deleted or destroyed by homologous recombination in a bacterium belonging to the genus Morella, a region adjacent to a gene encoding orotate phosphoribosyltransferase. A primer represented by SEQ ID NO: 1 and / or SEQ ID NO: 2 to be amplified.

(Claim 6)
The primer shown by sequence number 5 and / or sequence number 6 which amplifies only an adjacent area | region using the arrangement | sequence amplified by the primer of Claim 5 as a template.

(Claim 7)
A primer used in the production of an auxotrophic strain in which a gene on a chromosome is deleted or destroyed by homologous recombination in a bacterium belonging to the genus Morella, wherein the gene encoding orotate phosphoribosyltransferase is deleted or destroyed. The primer shown by sequence number 9 and / or sequence number 10 which confirms.

  According to the present invention, a bacterium belonging to the genus Morella that can be used for a wide range of uses as a host cell can be provided.

  In addition, according to the present invention, a primer that can be used when producing a bacterium belonging to the genus Morella that can be used as a host cell in a wide range of applications can be provided.

  That is, the bacterium belonging to the genus Morella of the present invention is an auxotrophic strain in which a homologous site and a deleted or destroyed gene are known.

  Using the bacterium belonging to the genus Morella of the present invention as a host cell, a gene having a gene deleted or destroyed between homologous sites or a plasmid having a useful gene is prepared and further homologous recombination is performed to produce an excellent strain. be able to.

  For example, if the useful gene is a gene involved in ethanol production, it can be used as a host cell to produce a strain with high ethanol productivity. By selecting useful genes according to the purpose, they can be used for a wide range of uses as host cells.

  Embodiments of the present invention will be described below.

  Homologous recombination techniques are used to delete or destroy genes on the chromosome.

  Homologous recombination is recombination that occurs in a portion having a homologous base sequence of a pair of double-stranded DNAs, and is one means for artificially changing a specific gene on the genome. When DNA having homology with a specific base sequence on the genome is introduced into a cell, recombination occurs at this homologous portion, and foreign DNA is incorporated into the genome. This can be used to destroy a specific gene or replace it with another gene.

  In addition, the introduction of genes by homologous recombination into genomic DNA has the problem that it is difficult to have multiple copies of the introduced gene compared to the method using a plasmid vector. Therefore, there is no need to continuously apply selective pressure such as adding an antibiotic to the medium, and there is an advantage that the character is stable.

For disruption of a gene by homologous recombination (double crossover), the upstream and downstream regions adjacent to the gene (may include part of the protein coding region of the gene) are cloned, and the adjacent upstream and downstream regions cloned. A plasmid (vector) is prepared, and host cells are transformed with this plasmid. The flanking sequence has at least 90%, preferably at least 95%, more preferably 98%, and even more preferably 99% homology to the corresponding sequence of the target cell genome. This plasmid must not contain the entire protein coding region of the gene, but may contain a portion of the protein coding region of the target gene. This adjacent region is at least 100 base pairs, and theoretically there is no upper limit on the length of the adjacent sequence, but preferably up to 4000 base pairs, and more preferably the adjacent region is 500 base pairs to 1200 base pairs long. That's it.

  Specific types of auxotrophic strains conferred by gene deletion or disruption are not particularly limited, and include, for example, leucine, histidine, methionine, arginine, tryptophan, lysine and other amino acid auxotrophs; uracil, Examples include nucleobase-requiring strains such as adenine; vitamin-requiring strains; among these, imparting uracil-requiring requirement is desirable for the following reasons.

  The uracil requirement can be obtained by disrupting a gene encoding an enzyme involved in the biosynthetic pathway of pyrimidine bases.

  The biosynthetic pathway of pyrimidine bases (thymine, cytosine, uracil) is composed of a common enzyme system from bacteria to fungi, plants and animals, and is an essential pathway for all organisms except a few pathogens . This pathway has been found to consist of six enzymatic reactions leading to UMP (uridine monophosphate), the precursor of all pyrimidine bases. 6 enzymes (and the gene that encodes them); The biosynthetic pathway of pyrimidine bases in E. coli and Salmonella typhimurium is shown in FIG. Strains in which the genes encoding these enzymes are disrupted cannot biosynthesize UMP, and thus become uracil-requiring strains.

Among these, for strains in which pyrE (gene encoding orotate phosphoribosyltransferase) or pyrF (gene encoding orotidine-5-phosphate decarboxylase) is disrupted, 5-fluoroorotic acid (5-FOA) It is known that positive selection by is possible. 5-FOA is incorporated into the pyrimidine pathway instead of orotate in strains without gene disruption, and finally incorporated into RNA to inhibit normal RNA synthesis and kill cells. The cells can survive because they are not metabolized in the disrupted strain and the pyrF disrupted strain. By utilizing this, by using a medium containing 5-FOA and a sufficient amount of uracil, it becomes possible to select and isolate a pyrE or pyrF-disrupted strain by the homologous recombination described above. If this pyrE or pyrF is used as a selection marker, it is considered that it can be surely expressed because it is a gene originally possessed by a bacterium, compared to a method of introducing an antibiotic resistance gene as a selection marker. For this reason, it is preferable to destroy the pyrE or pyrF gene for imparting uracil requirement.

  To delete or destroy the gene (pyrE) encoding orotate phosphoribosyltransferase by homologous recombination in a bacterium belonging to the genus Morella, (1) first clone pyrE and its upstream and downstream flanking regions to clone a plasmid (2) It is also necessary to prepare a plasmid from which the pyrE portion has been removed from the plasmid.

  For the cloning of (1) above, primers of SEQ ID NO: 1 and SEQ ID NO: 2 can be used. SEQ ID NO: 1 is a forward primer and SEQ ID NO: 2 is a reverse primer. SEQ ID NOs: 1 and 2 can amplify the pyrE gene and about 1000 bp adjacent upstream and downstream of the pyrE gene.

In order to remove only the pyrE gene part from the region amplified in (1) above, SEQ ID NO: 5 and / or
Alternatively, the primer of SEQ ID NO: 6 can be used. SEQ ID NO: 5 is a forward primer and SEQ ID NO: 6 is a reverse primer. SEQ ID NO: 5 was amplified in (1) in order to amplify from the sequence cloned in (1) to the ORF of the pyrE gene, and SEQ ID NO: 6 from the end of the pyrE gene ORF to the end of the downstream adjacent region. When PCR is performed using the region SEQ ID NO: 5 and 6 primers as a template, only the region excluding the pyrE gene is amplified.

  The primers of SEQ ID NOs: 9 and 10 can be used to confirm whether pyrE has been deleted or destroyed from the generated cells.

  The primers of the present invention are described in Moorella thermoacetia, M. et al. thermoautotrophica, M.M. For the production of a bacterium belonging to the genus Morella, such as glycerini, and a strain with high ethanol production efficiency, preferably a bacterium belonging to the genus Morella having an ability to produce ethanol from hydrogen and carbon dioxide or a synthesis gas of carbon monoxide, more preferably Moorella sp. It is a primer that can be used when a gene encoding HUC22-1 orotate phosphoribosyltransferase is deleted or destroyed by homologous recombination.

  The bacterium belonging to the genus Morella of the present invention is Moorella thermoacetia, M. p. thermoautotrophica, M.M. Nutritional requirements are given to Morella bacteria such as glycerini, and the ability to produce ethanol from hydrogen and carbon dioxide or carbon monoxide synthesis gas is particularly important in producing strains with high ethanol production efficiency. The bacterium belonging to the genus Morella has auxotrophy, and moreover, Moorella sp. What gave auxotrophy to HUC22-1 is preferable.

  Moorella sp. A strain that has disrupted the pyrE gene of HUC22-1 by homologous recombination to confer uracil requirement is Moorella sp. 22-1-U (reception number NITE AP-606) is deposited with the Patent Microorganism Depositary Center for Product Evaluation Technology.

  The culture properties are as follows.

In a modified ATCC 1754 PETC agar medium (* 1), anaerobic, circular colonies having a diameter of 1 to 2 mm are formed at 55 ° C. for 3 to 5 days of culture.
i) Color: White ii) Surface shape: Smooth iii) Transparency: Opaque

* 1 Modified ATCC 1754 PETC Agar NH ₄ Cl 1.0 g
KCl 0.1g
MgSO ₄ 0.2g
NaCl 0.8g
KH ₂ PO ₄ 0.1 g
CaCl ₂ 0.02 g
Yeast Extract 1.0g
(When Yeast Extract is not added, Uracil 0.01 g)
NaHCO ₃ 2.0 g
Cysteine-HCl 0.3g
Trace element solution (I) 10ml
Vitamin solution (II) 10ml
Distilled water 1000ml
(Agar (for high temperature) 20g)
(Fructose 5.0g)
pH 5.9 (before sterilization)
Sterilization temperature / time 121 ° C 15 minutes

  In addition, said (I) Trace element solution and (II) Vitamin solution are the following compositions.

(I) Trace element solution
Nitrilotrific acid 2.0g
MnSO ₄ · H ₂ O 1.0 g
_{Fe (SO 4) 2 (NH} 4) 2 · 6H 2 O 0.8g
CoCl ₂ · _{6H 2} O 0.2g
ZnSO ₄ · 7H ₂ O 0.0002 g
CuCl ₂ · 2H ₂ O 0.02 g
NiCl ₂ · _{6H 2} O 0.02g
_{Na 2 MoO 4 · 2H 2 O} 0.02g
Na ₂ SeO ₄ 0.02 g
Na ₂ WO ₄ 0.02 g
Distilled water 1000ml

(II) Vitamin solution
Biotin 2.0mg
Folic acid 2.0mg
Pyridoxine-HCl 10mg
Thiamine-HCl 5.0mg
Riboflavin 5.0mg
Nicotinic acid 5.0mg
D-Ca-pantothenate 5.0mg
Vitamin B ₁₂ 0.1mg
p-Aminobenzoic acid 5.0mg
Thiocic acid 5.0mg
Distilled water 1000ml

  This strain has uracil requirement because it lacks the pyrE gene encoding orotate phosphoribosyltransferase.

  It also produces acetic acid and ethanol from fructose. Acetic acid and ethanol are produced from carbon dioxide and hydrogen, or carbon monoxide and carbon dioxide.

  Since this strain has known homologous sites and disrupted genes, for example, when introducing a useful gene such as a gene that contributes to improvement in ethanol productivity and attempting to create a strain with excellent ethanol production ability, The Morella bacterium of the present invention can be used as a host cell.

  That is, a vector in which the pyrE gene and the gene to be introduced (useful gene) are inserted between two homologous sites is prepared and recombined to select the target gene by introduction and revival of uracil biosynthesis ability. Thus, further recombinant cells can be obtained.

  Examples of the present invention will be described below, but the present invention is not limited to such examples.

  It should be noted that Moorella sp. The strain is referred to as a pyrE disruption strain, and Mooreella sp. The HUC22-1 strain may be referred to as HUC22-1 or a wild strain.

Example 1
1. Moorella sp. Extraction of genomic DNA from HUC22-1 strain Moorella sp. For the extraction of genomic DNA from HUC22-1, the following method modified from the Marmur method (reference document 1) was used.

  20 ml of the culture solution cultured to the stationary phase using fructose as a substrate was collected by centrifugation at 3,000 rpm for 15 minutes, and the supernatant was removed. To the obtained cells, 1.5 ml of TEbuffer (10 mM Tris, 1 mM EDTA-2Na) and 5 mg of lysozyme (powder) were added and suspended, and the mixture was gently shaken at 37 ° C. for 30 minutes. Thereafter, 2.5 ml of lysate [100 mM Tris-HCl (pH 8.0), 300 mM NaCl, 20 mM EDTA-2Na, 2% SDS, 100 μg / ml Proteinase K] was added and mixed. The cells were allowed to stand for minutes to dissolve the cells. Add 1.25 ml of phenol: chloroform: isoamyl alcohol (25: 24: 1) mixture to the cell lysate and shake at room temperature for 10 minutes to thoroughly mix, then centrifuge at 11,000 rpm for 5 minutes to form 3 layers Separate and transfer the upper layer to a new tube. This operation was repeated twice in total to remove the protein. Add 1/10 volume of 3M sodium acetate solution (pH 5.2) and 2.5 volumes of ethanol, mix gently, let stand at room temperature for 10 minutes, then 11,000 rpm for 15 minutes. The supernatant was removed by centrifugation, and the DNA was precipitated. Thereafter, 5 ml of 75% ethanol was added and centrifuged at 11,000 rpm for 5 minutes to remove the supernatant and rinse the DNA. Finally, it was centrifuged at 11,000 rpm for 30 seconds to completely remove excess ethanol, dissolved in 200 μl of ultrapure water, and stored at −20 ° C. The obtained genomic DNA was diluted 10-fold with ultrapure water, and the concentration and purity were confirmed in the DNA quantification mode of a spectrophotometer (Ultrospec3000, Pharmacia Biotech).

2. Search of pyrE from the genome database of M. thermoacetica ATCC 39073 strain M. thermoacetica, a related species of HUC22-1 strain registered in Genbank
BLAST from the genome database (accession number: CP000232) of ATCC 39073 strain
Using the search, a gene considered to be pyrE was searched.

3. Cloning of PyrE derived from HUC22-1 strain genome PCR was performed using Premix Taq ^™ (TaKaRa Ex Taq ^™ Version) using the genomic DNA of HUC22-1 strain obtained in 1 above as a template, and pyrE found in 2 above. The gene considered to have been amplified including about 1,000 bp before and after ORF. The PCR reaction solution composition and reaction conditions are shown in Table 1, and the primer sequences used are shown in Table 2. After completion of the reaction, each PCR product was confirmed by 0.7% agarose gel electrophoresis.

  The result confirmed a single band of the expected size for pyrE.

Next, it was incorporated into pGEM ™ -T Easy vector by TA cloning using pGEM ™ -T Easy Vector System, introduced into E. coli DH5a by the heat shock method, and transformed. The cells were cultured overnight on an LB plate containing 100 μg / ml ampicillin, 0.1 mM IPTG, and 40 μg / ml X-gal, and colonies having a plasmid in which the target gene was cloned were selected by Blue / White selection. Each selected colony was inoculated into 2 ml of LB medium containing 100 μg / ml ampicillin and further cultured with shaking overnight, and then the plasmid was recovered using Quantum Prep ™ Plasmid Miniprep Kit. Each recovered plasmid was cleaved with an appropriate restriction enzyme, and it was confirmed that the target fragment was cloned by 0.7% agarose gel electrophoresis. Sequencing of the fragments cloned in each confirmed plasmid was commissioned to the DNA sequencing service of the Department of Genetics, Hiroshima University Natural Sciences Research Support and Development Center. First, the sequence was determined from both ends using the M13 forward primer and M13 reverse primer, and the inner sequence was determined using the primers shown in Table 3.

As a result, it was confirmed that the sequence has 99% or more homology with the sequence shown in the genome database of M. thermoacetica ATCC 39073 strain.

4). Construction of plasmid pGpyrE for destroying pyrE
According to the method of Sato et al. (Reference document 2), a plasmid for destroying pyrE by homologous recombination (double crossover) (FIG. 2) was constructed. An outline of the construction is shown in FIG. Adapter PCR was performed using Premix TaqTM (TaKaRaEx TaqTM Version) using the plasmid pGEMPyrE obtained in 3 above as a template to amplify the part excluding the ORF of pyrE on the plasmid, and a BamHI site was added to both ends thereof. . The PCR reaction solution composition and reaction conditions are shown in Table 4, and the primer sequences used are shown in Table 5. The obtained PCR product was confirmed by 0.7% agarose gel electrophoresis, then digested with restriction enzyme BamHI overnight, and purified using MagExtractor ™ -PCR & Gel Clean Up. Thereafter, ligation was performed using DNA Ligation Kit Ver. 2 to construct pyrE destruction plasmid pGDPyrE (FIG. 3).

5). Construction of plasmid pGDyrE2 for destroying pyrE In order to confirm whether transformation can be performed even if the homology region for causing double crossover is shortened, the previous and subsequent homology regions were determined by PCR from the pGDyrE constructed in 4 above. Fragments were taken out in increments of 500 bp, and plasmid pGDPyrE2 (FIG. 3) was re-cloned into pGEM ™ -TEasy vector (the cloning method was the same as in 3 above). The PCR reaction solution composition and reaction conditions are shown in Table 6, and the primer sequences used are shown in Table 7.

6). Disruption of pyrE on the HUC22-1 strain genome by homologous recombination Electroporation was used for gene introduction into the HUC22-1 strain. The procedure was as follows with reference to the method of Sato et al.

The medium and buffer used were all prepared anaerobically and aseptically, and all operations were performed anaerobically while blowing carbon dioxide gas. 20 ml of the culture solution of this bacterium cultured up to the late logarithmic growth phase (turbidity, wavelength 660 nm, OD ₆₆₀ = 1.5 to 2.0) using fructose as a substrate is centrifuged at 6,000 rpm at 4 ° C. for 5 minutes to remove the supernatant. The cells were collected. The cells were resuspended in 5 ml of 272 mM sucrose solution, washed by centrifugation at 6,000 rpm and 4 ° C. for 5 minutes, and the supernatant was removed. After this washing was repeated twice in total, the cells were resuspended in 1 ml of 272 mM sucrose solution. A solution containing 5 to 10 μg of DNA (pGDPyrE or pGDPyrE2) to be introduced into the cells was added and mixed. In the case of the negative control, the same amount of ultrapure water was added instead of the DNA solution. After leaving it in ice for 10 minutes, transfer 100 μl to a 2 mm gap electroporation cuvette (ELECTROPORATIONCUVETTES PLUSTM, BTX) that has been cooled in ice and set it in the electroporation apparatus (ECM 630, BTX). Then, a pulse was applied once under the conditions of 2.0 kV (10.0 kV / cm), 400Ω, and 50 μF. The pulsed solution is collected using a plastic syringe (Terumo) fitted with a needle (Nipro) and added to 2 ml fructose medium (containing 10 μg / ml uracil) in an 8 ml vial. It was. After standing in ice for 10 minutes, pre-culture was performed at 55 ° C. for 10 hours.

  The total amount of the preculture solution is added to a fructose medium containing 0.2% (w / v) 5-FOA, 10 μg / ml uracil, and 2% (w / v) high-temperature culture agar (Nacalai Tesque) as selective substances. In addition, a roll tube was produced. However, 5-FOA was not added to the positive control. The prepared roll tube was allowed to stand at 55 ° C. and cultured for 4 to 7 days. The obtained colony was anaerobically collected using a sterilized Pasteur pipette, and 0.2% (w / v) of 5- It was inoculated into fructose liquid medium containing FOA and 10 μg / ml uracil.

  After two passages, when part of the plant was subcultured onto a completely synthetic medium containing neither uracil nor yeast extract, the wild strains could grow without problems, but these strains could not grow at all.

  Moreover, when only 10 μg / ml uracil was added to the complete synthetic medium, these strains could grow. From these, it was confirmed that these strains were transformed into uracil-requiring strains (pyrE disrupted strains).

  The transformation efficiency was about 35 per μg of DNA when using pGDyrE, and about 8 per 1 μg of DNA when using pGDPyrE2.

7). Confirmation of destruction of pyrE by PCR PCR was carried out using Premix Taq ™ (TaKaRa Ex Taq ™ Version) using the genomic DNA of the wild strain and the pyrE disruption strain as a template to confirm the destruction of pyrE. The PCR reaction solution composition and reaction conditions are shown in Table 8, and the primer sequences used for each gene are shown in Table 9. After completion of the reaction, the size of each PCR product was confirmed by 0.7% agarose gel electrophoresis.

  As a result, it was confirmed that the band (about 2,600 bp) seen in the wild strain was lost.

  It was also found that in the pyrE disrupted strain, it is highly possible that the sequence is lost over a length of at least about 8 kbp upstream and at least 2 kbp downstream of pyrE.

[References]
(1) Marmur, J .: A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 1961; 3: 208-18

(2) Sato, T., Fukui, T., Atomi, H., and Imanaka, T .: Improved and versatile transformation system allowing multiple genetic manipulations of thehyperthermophilic archaeon Thermococcus kodakaraensis. Appl. Environ. Microbiol. 2005; 71: 3889- 99

Biosynthetic pathway of pyrimidine base Destruction of pyrE by homologous recombination (double crossover) Construction of plasmids for destroying pyrE, pGDPyrE and pGDPyrE2.

Claims (7)

  A bacterium belonging to the genus Morella, wherein a gene on a chromosome is deleted or destroyed by homologous recombination and exhibits auxotrophy.   The bacterium belonging to the genus Morella according to claim 1, wherein the gene on the chromosome is a gene encoding an enzyme involved in a biosynthetic pathway of a pyrimidine base, and the auxotrophy is uracil auxotrophy.   The bacterium belonging to the genus Morella according to claim 1 or 2, wherein the gene on the chromosome is a gene encoding orotate phosphoribosyltransferase.   Moorella sp. The bacterium belonging to the genus Morella according to any one of claims 1 to 3, which has been deposited at the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation, 22-1-U (reception number NITE AP-606).   A primer used in the production of an auxotrophic strain in which a gene on a chromosome has been deleted or destroyed by homologous recombination in a bacterium belonging to the genus Morella, a region adjacent to a gene encoding orotate phosphoribosyltransferase. A primer represented by SEQ ID NO: 1 and / or SEQ ID NO: 2 to be amplified.   The primer shown by sequence number 5 and / or sequence number 6 which amplifies only an adjacent area | region using the arrangement | sequence amplified by the primer of Claim 5 as a template.   A primer used in the production of an auxotrophic strain in which a gene on a chromosome is deleted or destroyed by homologous recombination in a bacterium belonging to the genus Morella, wherein the gene encoding orotate phosphoribosyltransferase is deleted or destroyed. The primer shown by sequence number 9 and / or sequence number 10 which confirms.

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