JP2017018061A - Method for producing transformant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a transformant of Clostridium cellulovorans, and a transformant of Clostridium cellulovorans produced by the production method.SOLUTION: The method comprises selecting an antibiotic capable of suppressing proliferation of Clostridium cellulovorans, and preparing a plasmid containing a resistance gene for the antibiotic. The method produces a transformant of Clostridium cellulovorans by the step of introducing the plasmid into Clostridium cellulovorans.SELECTED DRAWING: None

Description

  The present invention relates to a transformant of Clostridium cellulovorans and a method for producing the same.

  In the genus Clostridium, there is a butanol-producing clostridium genus that produces solvent such as butanol, acetone, ethanol, etc., such as Clostridium acetobutylicum and Clostridium beijerinckii. In addition, there exist cellulosome-producing Clostridium genera that can form a superprotein complex called cellulosome and decompose lignocellulose, such as Clostridium cellulovorans and Clostridium thermocellum.

Clostridium cellulovorans is a microorganism that can directly degrade soft biomass such as rice straw. In recent years, genome analysis and analysis of various properties of saccharide-degrading enzymes involved in biomass degradation have been carried out (Patent Document 1, non-patent document 1). Patent Documents 1 and 2).
The main metabolites of Clostridium cellulovorans are organic acids such as formic acid, acetic acid, butyric acid, and lactic acid. With existing technology, it is possible to produce raw materials for chemical products that have characteristics similar to petroleum plastics such as polylactic acid from lactic acid. Is possible.
Clostridium cellulovorans can produce lactic acid directly from renewable cellulosic biomass, but the production volume is low and the production is expected to increase by genetic modification.

In recent years, genome editing, which has been in the spotlight as a next-generation gene modification method, is rapidly spreading as a technology that can freely rewrite genome information regardless of cell type or organism type. In particular, the development of CRISPR / Cas9 (clustered regularly interspaced short linear repeats / CRISPR-associated protein 9), which is a third generation genome editing tool using RNA-induced nucleases, has markedly lowered the threshold of genome editing (CRISPR-associated protein 9). Non-patent document 3, see).
CRISPR / Cas9 is a part of the CRISPR / Cas system, which is an exogenous mechanism of exogenous DNA originally possessed by eubacteria and archaea, applied to genome editing. Research on gene editing using the CRISPR / Cas9 system has been widely reported, starting with human cells and mouse cells, to crickets and small fish (see Non-Patent Documents 4 and 5). As for bacteria originally possessed as an elimination mechanism, the use of the CRISPR / Cas9 system has been reported for some bacteria in recent years.

In order to perform gene modification of a target gene, a technique for introducing a gene into a host fungus, that is, a transformation method is necessary. Clostridium was first established as a butanol-producing bacterium, Clostridium acetobutylicum, and by Ultram et al., A shuttle of E. coli-C. Acetobutyricum, a host-vector system for clostridium acetobutylicum. A vector was prepared (see Non-Patent Document 6).
Thereafter, in order to give Clostridial acetobutylicum the ability to decompose cellulose, Clostridium cellulovolans-derived endo β-1,4-D-glucanase was expressed by Kim et al. (See Non-Patent Document 7). In addition, Minton et al. Developed a vector called ClosTron, which enabled transformation in several microorganisms belonging to the genus Clostridium, including Clostridium acetobutylicum (see Non-Patent Document 8).

  In Clostridium begerinki, which is the same butanol-producing bacterium, Lopez-Contreras et al. Succeeded in transforming fungal cellulase into Clostridium begerinki using a shuttle vector similar to clostridium acetobutylicum (see Non-Patent Document 9). . Furthermore, the Blaschek group has succeeded in genome editing by the CRISPR / Cas system in Clostridium Beigelinki (see Non-Patent Document 10).

  On the other hand, in the genus Clostridium producing cellulosome, a host-vector system is prepared by Lynd et al. In clostridium thermocellum, and a transformation method has been established (see Non-Patent Document 11). Subsequently, the transformation method was applied, and the destruction of the target gene of clostridium thermocellum was reported by Tripati et al. (See Non-Patent Document 12). From these circumstances, it can be expected that genetic modification is also performed in the cellulosome-producing Clostridium genus by establishing a transformation method.

  In Patent Document 2, as a method for replacing a DNA sequence for both butanol-producing Clostridium genus such as Clostridium acetobutylicum and cellulosome-producing Clostridium genus such as Clostridium thermocellum, replication that can be replicated in Clostridium bacteria A replacement cassette comprising a first marker gene sandwiched between two sequences homologous to a selected region around the origin and the target DNA sequence, a method of transformation with a vector comprising the second marker gene, etc. It is disclosed.

  However, in these conventional techniques, there has been no report on transforming Clostridium cellulovorans, and a host-vector system for the purpose of transforming Clostridium cellulovorans has not been established. Therefore, the inventors of the present application tried to construct a host-vector system for Clostridium cellulovolans and establish a transformation method.

International Publication No. 2010/53363 Pamphlet Patent No. 523375

Tamaru, Y. et al. , Miyake, H .; Kuroda, K .; , Nakanishi, A .; , Kawade, Y .; Yamamoto, K .; Uemura, M .; , Fujita, Y .; , Doi, R .; H. Ueda, M .; “Genome sequence of the cellulosome-producing mesophilic organic Clostridium cellulovorans 743B. Bacteriol. 192 (3), 901-902. (2010) Tamaru, Y. et al. , Miyake, H .; Kuroda, K .; Ueda, M .; , Doi, R .; H. “Comparative genomics of the mesophilic cellulosome-producing Clostridium cellulovorans and its application to biofuel production via conoside production. Technol. , 31 (8-9), 889-903. (2010) Le, C.I. , F.A. Ann, R.A. David, C .; , Shuailian, L .; , And Feng, Z. “Multiplex Genome Engineering Using CRISPR / Cas Systems.” Science 339, 819-823. (2013) Fujihara, Y. et al. , And Ikawa, M .; “CRISPR / Cas9-Based Genome Editing in Mice by Single Plasmid Injection.” Methods in Enzymology 546, 319-336. (2014) Ansai, S.A. , And Kinoshita, M .; “Targeted mutagenesis using CRISPR / Cas system in medaka.” Biologists 3,362-371. (2014) Ultram, J. et al. Loughlin, M .; Swinfield, TJ. Brehm, J .; K. Thompson, D .; E. Minton, N .; P. “Introduction of plasmids into whole cells of Clostridium acetobutylicum by electroporation.” FEMS Microbiol. Letters 56, 83-88. (1998) Kim, A.M. Y. , Attwood, G .; T. T. , Holt, S .; M.M. , White, B .; A. Blaschek, H .; P. “Heterologous expression of endo-β-1,4-D-glucanase from Clostridium cellulovorans in Clostridium acetobutylicum-3 ATCC 824 flooding transformation.” (1993) Heap, J. et al. T. T. Pennington, O. J. et al. , Cartman, S .; T. T. , And Minton, N .; P. "A modular system for Clostridium shuttle plasmids." J Microbiol Methods. 78 (1) 79-85. (2009) Lopez-Contreras, A.M. M.M. Smidt, H .; , Van der Ost, J. et al. Classen, P .; A. Mooibroek, H .; , And de Vos, W.M. M.M. “Clostridium beijerinckii cells expressing Neocallimastic patrickialum glycosides hydroloses show enhanced 51 ndroproduction. (2001) Wang, Y .; , Zhang Z. T. T. Seo, S .; O. , Choi, K .; Lu, T .; Jin, Y .; S. , And Blaschek, H .; P. “Markerless chromosomal gene deletion in Clostridium beijerinking using CRISPR / Cas9 system.” J Biotechnol. 200 1-5. (2015) Tyurin, M .; V. , Desai, S .; G. Lynd, L .; R. “Electrotransformation of Clostridium thermocellum.” Appl Environ Microbiol 70, 883-890. (2004) Tripati, S .; A. Olson, D .; G. Argyros, D .; A. Miller, B .; B. Barrett, T .; F. Murphy, D .; M.M. McCool, J .; D. Warner, A .; K. Raigaria, V .; B. Lynd, L .; R. Hogsett, D .; A. , Ciazza, N .; C. “Development of pyrF-based genetic system for targeted gene deletion in Clostridium thermocellum and creation of a caput mutant.” 65 Appl. (2010)

  An object of the present invention is to provide a method for producing a Clostridium cellulovorans transformant, and a Clostridium cellulovorans transformant produced by the method.

  The present inventors have intensively studied for the purpose of solving the above problems, selecting an antibiotic capable of suppressing the growth of Clostridium cellulovorans from various antibiotics, and preparing a plasmid containing a resistance gene of the antibiotic did. And it discovered that the transformant of Clostridium cellulovorans could be manufactured through the process of introduce | transducing this plasmid into Clostridium cellulovorans, and came to complete this invention.

That is, this invention relates to the method etc. which manufacture the transformant of Clostridium cellulovorans shown by following (1)-(6).
(1) A method for producing a transformant of Clostridium cellulovorans comprising the step of introducing a plasmid containing an antibiotic resistance gene capable of suppressing the growth of Clostridium cellulovorans into Clostridium cellulovorans.
(2) Clostridium The method according to (1) above, wherein the antibiotic capable of suppressing the growth of cellulovorans is at least one selected from chloramphenicol, spectinomycin, and thianphenicol.
(3) Clostridium The method according to (1) or (2) above, wherein the antibiotic resistance gene capable of suppressing the growth of cerulovorans is derived from the genus Clostridium.
(4) The method according to any one of (1) to (3) above, wherein the antibiotic resistance gene capable of suppressing the growth of Clostridium cellulovorans is a chloramphenicol resistance gene shown in SEQ ID NO: 1 in Sequence Listing.
(5) A transformant of Clostridium cellulovorans produced by the method according to any one of (1) to (4) above.
(6) Clostridium A plasmid containing an antibiotic resistance gene capable of suppressing the growth of Clostridium cellulovorans, which is used for cellulovorans transformation.

  According to the present invention, a transformant of Clostridium cellulovolans can be produced. Since Clostridium cellulovorans can directly saccharify cellulosic biomass, the present invention makes it possible to modify metabolism-related enzymes and the like by introducing a target gene into Clostridium cellulovorans. This makes it possible to directly produce compounds such as butanol and succinic acid that are useful as raw materials for biofuels and chemical products from cellulosic biomass.

It is the figure which showed the state of the proliferation of the Clostridium cellulovorans in the chloramphenicol addition culture medium (Example 1). BRIEF DESCRIPTION OF THE DRAWINGS It is the figure which showed the outline | summary of the plasmid pMTL500C for Clostridium cellulovorans (Example 1). It is the figure which confirmed the detection of the colony of the transformant of the Clostridium cellulovorans in the chloramphenicol addition culture medium (Example 1). (Example 1) which is the figure which confirmed the detection of the chloramphenicol resistance gene in the transformant of Clostridium cellulovorans.

The “method for producing a transformant of Clostridium cellulovorans” of the present invention refers to a method of obtaining a transformant by carrying out transformation of Clostridium cellulovolans by introducing a foreign gene or the like into Clostridium cellulovorans.
This method essentially includes the step of introducing a plasmid containing an antibiotic resistance gene capable of suppressing the growth of Clostridium cellulovorans into Clostridium cellulovorans. The “method for producing a transformant of Clostridium cellulovorans” of the present invention includes, for example, a step of introducing a target gene into the plasmid and then introducing the plasmid into Clostridium cellulovorans. Other methods useful for the production of the transformant may be included.

Here, “an antibiotic capable of suppressing the growth of Clostridium cellulovorans” means that the growth of Clostridium cellulovorans in a growth environment where these antibiotics are present is suppressed compared to a growth environment where these antibiotics are not present. It refers to antibiotics that show action.
Such antibiotics include chloramphenicol, spectinomycin, thianphenicol, and the like. Any one or more of these or a combination of two or more may be used as the antibiotic of the present invention.

The resistance gene of these antibiotics may be any conventionally known resistance gene as long as it can be used as a selection marker for a transformant of Clostridium cellulovorans, but it is derived from the genus Clostridium. It is preferable that the gene is a resistance gene.
For example, in the case of a chloramphenicol resistance gene, it is preferable to use a chloramphenicol resistance gene derived from Clostridium perfringens as shown in SEQ ID NO: 1 in the sequence listing.

  The “plasmid containing an antibiotic resistance gene capable of suppressing the growth of Clostridium cellulovorans” of the present invention is a plasmid in which an antibiotic resistance gene as described above is incorporated into an expression vector useful for transformation of Clostridium cellulovorans. That means. As such an expression vector, any conventionally known expression vector can be used. For example, pMTL500E (see Non-Patent Document 6), which is a shuttle vector for Escherichia coli-Clostridial acetobutylicum, and the like are used. You can also.

The “plasmid containing an antibiotic resistance gene capable of inhibiting the growth of Clostridium cellulovorans” of the present invention is, for example, an antibiotic resistance gene that does not inhibit the growth of Clostridium cellulovorans is removed from pMTL500E, and the growth of Clostridium cellulovorans is suppressed there. It can be produced by incorporating a possible antibiotic resistance gene.
Examples of such a plasmid include pMTL500C in which the erythromycin resistance gene is removed from pMTL500E and the chloramphenicol resistance gene shown in SEQ ID NO: 1 is incorporated therein.

  The “transformant of Clostridium cellulovorans” of the present invention refers to a transformant of Clostridium cellulovolans produced by the method for producing a transformant of Clostridium cellulovorans of the present invention.

  Hereinafter, the present invention will be described more specifically with reference to examples, but it goes without saying that the present invention is not limited thereto.

[Example 1]
1. Examination of antibiotic resistance of Clostridium cerulovorans From several antibiotic resistance genes, the most suitable selection marker was examined as a marker for screening for genetic recombination in the production of the transformants of Clostridium cerulovorans.

1) Sample (1) Host cell Clostridium cellulovorans 743B (ATCC 35296) was used as a host cell.
In culturing this host cell, 3 g / L cellobiose, 4 g / L yeast extract, 12 g / L NaCl, various trace elements were phosphate buffered as a carbon source with reference to the method of Sleat et al. A medium was prepared by dissolving in the liquid.
10 mL of the produced medium was put into a 16 × 125 mm test tube for anaerobic culture, carbon dioxide was blown in for about 3 minutes using an anaerobic culture apparatus (Sangen Industrial Co., Ltd.), and the inside of the test tube was replaced with carbon dioxide. Thereafter, a butyl rubber stopper and a screw cap were applied, and autoclaved at 121 ° C. for 40 minutes for sterilization.
Clostridial cellulovorans were also inoculated using an anaerobic culture apparatus. The inside of the syringe of 1 mL volume was replaced with carbon dioxide from the carbon dioxide launcher, and the needle was inserted into the test tube containing the inoculum in a state filled with 0.2 mL of carbon dioxide, and the carbon dioxide was discharged into the test tube. Thereafter, 0.2 mL of the inoculum was taken out, rapidly inoculated into the prepared test tube medium, and cultured at 37 ° C.
Reference 1: Sleat, R .; , Mah, R .; A. , And Robinson, R .; “Isolation and Characterization of an Anaerobic, Cellulolytic Bacterium, Clostridium cellulovorans sp. Nov.” Apple Environ Microbiol 48, 88-93. (1984)

(2) Antibiotics Chloramphenicol, erythromycin, spectinomycin or thianphenicol (all Wako Pure Chemical Industries, Ltd.) were used as antibiotics. Of these, chloramphenicol and erythromycin were dissolved in 99.5% ethanol and stored frozen until use. Spectinomycin and thiamphenicol were dissolved in distilled water and stored frozen until use.

2) Culture of Clostridium Cellulovorans The antibiotic stock solutions prepared in 1) and (2) above were thawed at the time of use and added to 10 mL of liquid medium in an anaerobic chamber to the concentration shown in Table 1. . Thereafter, 0.2 mL of the clostridium cellulovorans culture solution cultured in (1) above was inoculated and cultured at 37 ° C.
For chloramphenicol, add it to the liquid medium so that the final concentration in the medium is 1 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, or 50 μg / mL. Culture was performed.

3) Results As a result of culturing under antibiotic resistance, Clostridium cellulovorans could not grow in a liquid medium supplemented with chloramphenicol, spectinomycin or thiamphenicol. Therefore, from these results, it was speculated that Clostridium cellulovorans did not retain the antibiotic resistance of chloramphenicol, spectinomycin or thiamphenicol.
Among these, in the liquid medium supplemented with chloramphenicol, as shown in FIG. 1, Clostridium cellulovorans could not grow even at low concentrations of 10 μg / mL and 15 μg / mL (FIG. 1, chloram). Amount of added phenicol, A: 1 μg / mL, B: 10 μg / mL, C: 15 μg / mL).

2. Construction of Clostridium cellulovorans plasmid From the results, chloramphenicol was selected from chloramphenicol, spectinomycin or thianphenicol, which are antibiotics that inhibit the growth of clostridial cellulovorans, and this was selected for screening for clostridial cellulovorans transformants. I decided to use it.

1) Sample (1) pMTL500E
PMTL500E, which is a shuttle vector for Escherichia coli-Clostridial acetobutylicum containing an ampicillin resistance gene and an erythromycin resistance gene as selection markers, was used as the base of a plasmid for Clostridium cellulovorans. Escherichia coli was used for amplification of pMTL500E, and selection with ampicillin was performed.

(2) Chloramphenicol resistance gene The chloramphenicol resistance gene was cloned from pJIR418 (see Reference 2), which contains the chloramphenicol resistance gene of Clostridium perfringens.
The nucleotide sequence of the chloramphenicol resistance gene is shown in SEQ ID NO: 1 in the sequence listing.
Reference 2: Sloan, J. et al. Warner, T .; A. , ScottP. T.A. , Bannam, T .; L. , Berryman, D .; , And Road JI. “Construction of a sequenced Clostridium perfringens-Escherichia coli shuffle plasmid.” Plasmid. 27, 207-219. (1992)

2) Construction of Clostridium Cellulovorans Plasmid The erythromycin resistance gene contained in pMTL500E was replaced with a chloramphenicol resistance gene to construct a clostridium cellulovorans plasmid.
That is, a linearized vector containing a region other than the erythromycin resistance gene of pMTL500E was prepared by PCR. At this time, a primer was designed so that a restriction enzyme site of PmeI and FseI was added to the 5 ′ end and 3 ′ end to prepare a linearized vector.
In addition, primers were designed from pJIR418 (see Reference 2) to add restriction sites for PmeI and FseI, and the chloramphenicol resistance gene including the upstream promoter region was amplified.
These pMTL500E linearized vectors and the chloramphenicol resistance gene were restricted with PmeI and FseI, and each gene was fractionated on an agarose gel and purified.
The purified DNA fragment and the linearized vector were subjected to a ligation reaction overnight at 16 ° C. using Ligation high (TOYOBO). Thereafter, the ligation product was transformed into E. coli DH5α strain, inoculated on an LB agar medium containing 100 μg / mL ampicillin, and cultured at 37 ° C. overnight.

Colony PCR was performed to confirm whether the chloramphenicol resistance gene, which is a selection marker, was inserted into the colony generated on the LB agar medium, and chloramphenicol was confirmed by confirming the PCR product using agarose electrophoresis. Colonies into which the resistance gene was inserted were selected. The colony was cultured in an LB liquid medium, and then plasmid DNA was extracted, and this Clostridium cellulovorans plasmid was designated as pMTL500C (FIG. 2). Ap ^{R in} FIG. 2 represents an ampicillin resistance gene, and Cm ^R represents a chloramphenicol resistance gene derived from Clostridium perfringens.

3. Method for Producing Transformant pMTL500C produced through the above steps 1 and 2 was transformed into Clostridium cellulovorans 743B (ATCC 35296) by electroporation.
That is, 10 mL of a clostridium cellulovorans 743B (ATCC 35296) culture solution was placed in a 5 mL tube and centrifuged for 5 minutes. Thereafter, the supernatant was removed, and the electroporation buffer (270 mM sucrose, 10 mM MgCl ₂ , 0.6 mM Na ₂ HPO ₄ , 4.4 mM NaH ₂ PO ₄ , pH 6.0) cooled against the precipitated cells. 1 mL of was added to suspend the cells, and centrifuged again for 5 minutes. The supernatant was again removed, 0.2 mL of a cold electroporation buffer not containing 10 mM MgCl ₂ was added and suspended, and the mixture was allowed to stand on ice for 10 minutes.

20 μL of the cell suspension and 440 ng of pMTL500C were added to a sterilized 1.5 mL Eppendorf tube and allowed to stand on ice for 10 minutes. Thereafter, 20 μL of this mixed solution was transferred to a 0.1 cm sterilized electroporation cuvette (BIO-RAD) cooled on ice, and the mixed solution was poured into the bottom of the cuvette to wipe off water droplets outside the cuvette.
The cuvette was installed in a Gene Pulser Xcell (registered trademark) electroporation system (BIO-RAD), and transformation was performed under conditions of 600 V, 200Ω, and 25 μF. After transformation, the whole cuvette was allowed to stand on ice for 10 minutes. Thereafter, 170 μL of medium was added, mixed, transferred to a 2 mL cryotube, and statically cultured at 37 ° C. for 2 hours. This culture solution was mixed by inversion to suspend the bacterial cells, and then the turbidity was measured with the culture solution in the test tube. When OD590 = 0.5 was reached, the test tube cultured was placed in the anaerobic chamber. .

  The cryotube containing the culture solution was taken out from the anaerobic chamber, and a medium containing 1.5% Agar was once dissolved in 7 mL of the medium. While blowing carbon dioxide from the anaerobic culture apparatus into the medium, chloramphenicol is added to the medium so that the final concentration is 15 μg / mL, and then the culture solution in the cryotube is quickly added while blowing carbon dioxide. Inoculated. A butyl stopper was applied, and a roll tube was produced with a roll tube creator (Sangen Industrial Co., Ltd.). The produced roll tube was statically cultured at 37 ° C. As a result, colonies could be detected 4 days after the start of culture (FIG. 3, right, circled area).

Colony direct PCR was performed in order to confirm whether the target gene was inserted into the colony generated in the roll tube. Primers were designed in a region containing chloramphenicol, and PCR was performed using colonies as templates. As a result, as shown in FIG. 4, a chloramphenicol resistance gene could be detected.
In FIG. 4, LaneM indicates 100 bp DNA Ladder (Bio Labs), Lane1 indicates wild-type Clostridium cellulovorans (negative control), Lane2 indicates pMTL500C transformant Clostridium cellulovorans, and Lane3 indicates pMTL500C (positive control) .

  Therefore, from this result, it was possible to produce a Clostridial cellulovorans transformant by using a plasmid containing the base sequence of the chloramphenicol resistance gene, which is used for transformation of Clostridium cellulovorans.

  According to the present invention, a transformant of Clostridium cellulovolans can be produced. According to the present invention, it is possible to modify a metabolism-related enzyme or the like by introducing a target gene into Clostridium cellulovorans. This makes it possible to directly produce compounds such as butanol and succinic acid that are useful as raw materials for biofuels and chemical products from cellulosic biomass.

Claims (6)

A method for producing a transformant of Clostridium cellulovorans comprising the step of introducing a plasmid containing an antibiotic resistance gene capable of suppressing the growth of Clostridium cellulovorans into Clostridium cellulovorans. The method according to claim 1, wherein the antibiotic capable of inhibiting the growth of Clostridium cellulovorans is at least one selected from chloramphenicol, spectinomycin and thianphenicol. The method according to claim 1 or 2, wherein the resistance gene of the antibiotic capable of suppressing the growth of Clostridium cellulovorans is derived from the genus Clostridium. 4. The method according to any one of claims 1 to 3, wherein the antibiotic resistance gene capable of suppressing the growth of Clostridium cellulovorans is the chloramphenicol resistance gene shown in SEQ ID NO: 1 in Sequence Listing. A transformant of Clostridium cellulovorans produced by the method according to claim 1. A plasmid containing an antibiotic resistance gene capable of suppressing the growth of Clostridium cellulovorans used for transformation of Clostridium cellulovorans.