阅读说明：本技术 革兰氏阳性菌的转化方法 (Transformation method of gram-positive bacteria ) 是由 吉田健一 于 2018-12-07 设计创作，主要内容包括：本发明的目的在于提供能将大尺寸的DNA无损伤地导入革兰氏阳性菌的宿主DNA中、简便且有效的革兰氏阳性菌的新型转化方法。进而,目的还在于提供,将期望的DNA片段蓄积于受体(受体菌)的染色体中,能够制作经人为设计的长的DNA的方法、转化后的细胞从控制自然环境的观点出发也不会成为问题的方法。本发明为通过接合转移进行的革兰氏阳性菌的转化方法,其特征在于,使用使DNA转移起始位点(oriT)区域失活的辅助质粒。上述辅助质粒优选为从pLS20cat使oriT区域失活的质粒。进而,优选为如下转化方法,所述方法中,供体菌具有：使上述DNA转移起始位点(oriT)区域失活的辅助质粒、及组入了上述DNA转移起始位点(oriT)区域的染色体DNA或质粒,且转移至受体菌。(The present invention is directed to a method for transforming a gram-positive bacterium, which is capable of accumulating a desired DNA fragment in the chromosome of a recipient (recipient bacterium) and which is capable of producing an artificially designed long DNA, and a method for transferring a transformed cell, which is not problematic from the viewpoint of controlling the natural environment.)

1. A method for transforming gram-positive bacteria by conjugative transfer, which comprises using a helper plasmid that inactivates the oriT region of the DNA transfer initiation site (oriT).

2. The transformation method according to claim 1, wherein the helper plasmid is a plasmid that inactivates the DNA origin of transfer (oriT) region from p L S20 cat.

3. The transformation method according to claim 1 or 2, wherein the donor bacterium in the conjugal transfer has: a helper plasmid for inactivating the region of the origin of DNA transfer (oriT), and a chromosomal DNA or plasmid into which the region of the origin of DNA transfer (oriT) is incorporated.

4. The transformation method according to claim 3, wherein the donor bacterium is at least one selected from the group consisting of gram-positive bacteria.

5. The transformation method according to claim 3 or 4, wherein the recipient bacterium is at least one selected from the group consisting of gram-positive bacteria.

6. The transformation method according to claim 5, wherein the donor bacterium and the recipient bacterium are Bacillus subtilis.

7. A method for transforming a gram-positive bacterium, comprising the steps of:

a step (1) for preparing a donor bacterium having: a helper plasmid for inactivating the region of the origin of DNA transfer (oriT), and a chromosomal DNA or plasmid into which the region of the origin of DNA transfer (oriT) is incorporated; and

and (2) performing a conjugative transfer of a recipient bacterium from the donor bacterium.

8. The transformation method according to claim 7, wherein the steps (1) and (2) are repeated using the transformed cell obtained by the conjugative transfer as a recipient bacterium, thereby accumulating a desired DNA fragment in a chromosome of the recipient bacterium.

Technical Field

The present invention relates to a method for transforming gram-positive bacteria.

Background

Conventionally, the following methods have been known as methods for transforming bacteria and the like: a competent cell transformation method in which a recombinant plasmid or a DNA fragment is introduced into a host such as Escherichia coli (see non-patent document 1); protoplast transformation, Electroporation (Electroporation), Agrobacterium mediated method, particle gun method, calcium phosphate method, lipofection method, and the like. However, in such a conventional transformation method, it is difficult to introduce a large-sized DNA fragment of, for example, 100kb or more into a host DNA.

As a method for introducing a large-sized DNA fragment into a host DNA, a method using conjugal transfer based on Bacillus subtilis which has acquired a natural transforming ability is known. However, this method has the following drawbacks: requiring a special culture condition for introducing Bacillus subtilis into cells having high natural transformation ability; the transformation itself takes time and effort. Furthermore, it is difficult to introduce a large-sized DNA exceeding several hundred kb into a host DNA without any damage.

On the other hand, it is known that the plasmid p L S20 derived from Bacillus natto has the ability to be transferred from gram-positive bacterial cells such as Bacillus subtilis to other gram-positive bacterial cells by conjugative transfer, and it has been clarified that this p L S20 functions as a helper plasmid in a donor and can transfer another plasmid possessed by the donor to a recipient (see non-patent documents 2 and 3). since p L S20, which is a helper plasmid together with another plasmid, is also transferred to a recipient-side gram-positive bacteria, conjugative transfer can be further repeated as a donor for other gram-positive bacteria, and this may cause a problem from the viewpoint of controlling the natural environment (see non-patent documents 4 and 5).

Disclosure of Invention

Problems to be solved by the invention

Under such circumstances, an object of the present invention is to provide a novel method for transforming gram-positive bacteria, which is capable of introducing large-sized DNA into a host DNA of gram-positive bacteria without causing damage and which is simple and effective. It is another object of the present invention to provide a method for accumulating a desired DNA fragment in a chromosome of a recipient (recipient bacterium) to produce an artificially designed long DNA, and a method for controlling the natural environment of a transformed cell without causing any problem.

Means for solving the problems

As a result of intensive studies to solve the above problems, the present inventors have found that p L S20cat can be obtained by using oriT_LS20The method of the present invention allows the transformed cell obtained to receive the conjugative transfer of the desired chromosome or plasmid as a recipient (recipient bacterium) by the same method again because the transformed cell does not contain the helper plasmid, and thus, the desired DNA fragment can be accumulated in the chromosome of the recipient (recipient bacterium) to design and produce a long DNAThe mixing of the liquid culture medium can be performed quickly and easily, and can be suitably used for the production of microorganisms, cultured cells, and the like in the industrial activities of synthetic biology. That is, the gist of the present invention is as follows.

[1] A method for transforming a gram-positive bacterium by conjugative transfer, characterized in that a helper plasmid is used which inactivates the region of the origin of DNA transfer (oriT).

[2] The transformation method according to [1], wherein the helper plasmid is a plasmid in which the oriT region is inactivated from p L S20 cat.

[3] The transformation method according to [1] or [2], wherein the donor bacterium in the conjugal transfer has: a helper plasmid for inactivating the region of the origin of DNA transfer (oriT), and a chromosomal DNA or plasmid into which the region of the origin of DNA transfer (oriT) is incorporated.

[4] The transformation method according to [3], wherein the donor bacterium is at least one selected from the group consisting of gram-positive bacteria.

[5] The transformation method according to [3] or [4], wherein the recipient bacterium in the conjugative transfer is at least one selected from the group consisting of gram-positive bacteria.

[6] The transformation method according to [5], wherein the donor bacterium and the recipient bacterium are Bacillus subtilis.

[7] A method for transforming a gram-positive bacterium, comprising the steps of:

a step (1) for preparing a donor bacterium having: a helper plasmid in which the region of the origin of DNA transfer (oriT) is inactivated, and a chromosomal DNA or plasmid in which the region of the origin of DNA transfer (oriT) is incorporated; and

and (2) performing a conjugative transfer of the recipient bacterium from the donor bacterium.

[8] The transformation method according to [7], wherein the desired DNA fragment is accumulated in the chromosome of the recipient bacterium by repeating the steps (1) and (2) using the transformed cell obtained by the conjugative transfer as the recipient bacterium.

[9] A gram-positive bacterium which comprises a helper plasmid for inactivating an oriT region and a chromosomal DNA or plasmid into which the oriT region has been incorporated.

[10] A helper plasmid with inactivated DNA transfer initiation site (oriT) region for the transformation of gram-positive bacteria by conjugative transfer.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the method for transforming gram-positive bacteria of the present invention, large-sized DNA can be transferred to the receptor side without any damage and in a short time, and oriT of p L S20cat is used_LS20According to the method of the present invention, since the obtained transformed cell does not have a helper plasmid, it is possible to receive the conjugative transfer of the desired chromosome or plasmid as a recipient (recipient bacterium) by the same method again.

Drawings

FIG. 1 is a diagram schematically showing the structure of the p L S20cat Δ oriT plasmid.

FIG. 2 is oriT_LS20Schematic representation of the loci and the inclusion of the kanamycin resistance gene of the donor strains.

FIG. 3 is a graph showing the efficiency of introduction of the transfer plasmid pGR16B, the helper plasmids p L S20cat and p L S20cat Δ oriT.

FIG. 4 is a graph showing the efficiency of introduction of chromosomal DNA, helper plasmids p L S20cat and p L S20 cat. DELTA. oriT.

FIG. 5 is a graph showing the efficiency of introduction of chromosomal DNA, helper plasmids p L S20cat and p L S20 cat. DELTA. oriT.

FIG. 6 is a comparison of p L S20cat and p L S20cat Δ oriT in the transfer of pGK1 into GK.

Detailed Description

The method for transforming gram-positive bacteria of the present invention will be described in detail below. In the present specification, unless otherwise specified, molecular biological methods such as preparation of DNA and vectors can be performed by methods generally described in the specification or methods based on the methods, which are well known to those skilled in the art. In addition, terms used in the present specification are to be interpreted according to the meanings commonly used in the technical field unless otherwise specified.

< transformation method of gram-positive bacterium >

The method for transforming a gram-positive bacterium of the present invention is characterized by using a helper plasmid that inactivates the DNA transfer initiation site (oriT) region in conjugal transfer.

Gram-positive bacteria in the present invention generally have a relatively thick (15nm to 80nm) cell wall, and most of them lack lipopolysaccharide on the outer layer, and bacteria having high sensitivity to lysozyme are more.

The transformation in the invention means that: modifications caused by the modification of the genomic DNA of gram-positive bacteria or the introduction of DNA into gram-positive bacteria include all trait changes caused by the deletion, replication, mutation of chromosomal DNA or the introduction of autonomously replicating plasmids. The introduced DNA may be incorporated into a chromosome for maintenance and replication, or may be maintained and replicated independently of a chromosome as in a plasmid or the like. Homologous recombination techniques may also be used when the DNA is incorporated into a particular locus of a gram-positive chromosome.

The term "conjugative transfer" as used herein refers to a phenomenon that occurs when bacteria having different properties are mixed and cultured, and means that a part of genes of a certain bacterium (donor bacterium) is transferred to another bacterium (recipient bacterium). The strength of the conjugative transfer ability is affected by the gene associated with the conjugative transfer on the chromosome of the donor or on the plasmid possessed by the donor. Genes involved in conjugative transfer include self-transmissible genes, conjugative transmissible genes, and oriT sequences. The protein encoded by the self-transmissible gene is involved in the interaction between the donor/recipient bacteria. The protein encoded by the conjugate transmissible gene has: the function of adding a notch to the oriT sequence and the function of stably transporting DNA that is single-stranded. The oriT sequence consists of a nick site and a recognition sequence for entry into the nick. These 3 coexists and the first splice transfer occurs.

In the present invention, the helper plasmid is a plasmid which is useful for gene transfer to a target bacterium when a certain gene is transferred to the bacterium, and specifically, p L S20 in Bacillus subtilis and the like are mentioned.

The DNA transfer initiation site (oriT) region in the present invention refers to a region containing a promoter sequence for DNA migration.

The method for transforming gram-positive bacteria of the present invention will be specifically described below. That is, the method for transforming gram-positive bacteria of the present invention comprises the steps of:

step (1): preparing a donor bacterium having: a helper plasmid in which the region of the origin of DNA transfer (oriT) is inactivated, and a chromosomal DNA or plasmid in which the region of the origin of DNA transfer (oriT) is incorporated;

step (2): the donor bacterium is used to perform conjugative transfer to a recipient bacterium.

The method for transforming gram-positive bacteria of the present invention preferably further comprises, if necessary, the step (3): the desired DNA fragment is accumulated in the chromosome of the recipient bacterium.

[ Process (1) ]

(Process for constructing recipient Strain)

As the recipient strain (recipient bacterium), Bacillus is preferable as described above, among them, Bacillus subtilis, Bacillus anthracis and thermophilic bacteria can be more preferably used, and Bacillus subtilis is further preferably used. In order to confirm the presence or absence of transformation by conjugative transfer in step (2), an antibiotic resistance gene was previously introduced into these recipient strains (recipient bacteria). Introduction of an antibiotic resistance gene into a recipient strain (recipient bacterium) can be carried out by a method known in the art.

For example, when Bacillus subtilis is used as a recipient strain (recipient bacterium), the Bacillus subtilis gene, such as comK, can be introduced by inactivating the gene by substitution with a spectinomycin resistance gene or the like. That is, 2 DNA fragments corresponding to the upstream and downstream regions of comK et al gene, respectively, were amplified by PCR using Bacillus subtilis DNA as a template. At this time, primers suitable for the upstream fragment and the downstream fragment were designed and used. In addition, other DNA fragments containing the spectinomycin resistance gene and the like of other Bacillus subtilis strains such as TMO310 strain were amplified using appropriate primers. The 3 fragments were ligated by recombinant PCR, and a spectinomycin resistance gene or the like was inserted between the upstream and downstream regions of comK or the like. A novel Bacillus subtilis strain modified by the recipient strain of the present invention can be obtained by transforming Bacillus subtilis into a recombinant PCR fragment and introducing the fragment into the recipient strain to impart resistance to antibiotics such as spectinomycin.

(construction of helper plasmid for inactivating oriT region)

The inactivation of the oriT region of the helper plasmid can be carried out by a conventional gene manipulation method known to those skilled in the art, and includes deletion of a part of the oriT region, deletion of the entire oriT region, and substitution of 1 or more bases of the oriT region, and for example, deletion of the oriT region by a method such as scarless deletion (marker-free deletion) can be used to inactivate the oriT region, and examples using p L S20cat as the helper plasmid will be described below.

The oriT corresponding to p L S20cat was amplified by PCR using p L S20cat DNA as a template and primers oriT-uF/oriT-uR and oriT-dF/ori in the upstream region_LS202 DNA fragments of the upstream (fragment 1) and downstream (fragment 2) regions of (a). The end of fragment 1 and the front end of fragment 2 are identical at 22bp, and therefore oriT can be converted by using these regions_LS20The region is missing. Another DNA fragment of the mazF kan cassette (fragment 3) was amplified from TMO311 DNA using mazF-F/mazF-R as primers. The 3 PCR fragments can be designed by ligation in the order of 1-3-2 by recombinant PCR using oriT-uF/oriT-dR as primers. Next, the recombinant PCR fragment was transformed into PKS11 strain and kanamycin resistance was imparted to the resulting strain, thereby obtaining a novel strain YNB 022. The strain can be obtained by oriT_LS20Region double-crossover events were incorporated into PCR fragments to modify p L S20cat, YNB022 at 37 ℃ for around evening propagation in L B liquid medium containing kanamycin, aliquots of the culture (aliquot) were transferred to fresh L B liquid medium containing 1mM Isopropylthiogalactopyranoside (IPTG), cells were propagated at 37 ℃ for around 2 hours, secondly, aliquots of the culture were spread on L B well plates containing 1mM IPTG, incubated at 37 ℃ for around one hour_LS20The region is accurately deleted. The primers were those shown in Table 2 below.

(construction procedure of Donor Strain)

As the donor strain (donor bacterium), Bacillus is preferable as described above, among them, Bacillus subtilis, Bacillus anthracis and thermophilic bacteria can be more preferably used, and Bacillus subtilis is further preferably used. In this step, in order to produce a donor strain, Bacillus subtilis into which a helper plasmid having the oriT region prepared as described above inactivated has been introduced in advance is used, and the oriT region derived from the helper plasmid is incorporated into the chromosomal DNA. Alternatively, a plasmid may be transferred instead of the chromosomal DNA, and in this case, a plasmid containing the oriT region derived from the helper plasmid is introduced into Bacillus subtilis into which a helper plasmid for inactivating the oriT region has been previously introduced, thereby preparing a donor strain that can be used in step (2).

The procedure for incorporating the oriT region derived from the helper plasmid into the chromosomal DNA of the donor strain can be carried out by a method known in the art by genetic manipulation by those skilled in the art, and the following will describe the procedure in the case of constructing Bacillus subtilis YNB060 as a donor strain using p L S20cat as the helper plasmid.

That is, 2 fragments corresponding to the upstream (fragment 1) and downstream (fragment 4) regions of yhfM were amplified from Bacillus subtilis strain 168DNA using primers yhfM-uF/yhfM-uR1 (for upstream) and yhfM-dF/yhfM-dR (for downstream). oriT-containing oriT was amplified using primers oriT-F/oriT-R and p L S20cat as a template_LS20Fragment 2 of (1). Further, fragment 3 having an erythromycin resistance gene was amplified using primer erm-F1/erm-R and plasmid pMutin2 as a template. Fragments 1 to 4 were ligated in the order of 1-2-3-4 by recombinant PCR using the primer yhfM-uF/yhfM-dR. The strain TMO311 (aprE:: kan) can be transformed by recombinant PCR fragments, and colonies resistant to both erythromycin and kanamycin are selected as the donor strain (YNB 060).

[ Process (2) ]

In this step, the recipient strain was transferred by conjugation from the donor strain, the donor strain and the recipient strain were cultured at about 37 ℃ in L B liquid media each containing an appropriate antibiotic while shaking at 180rpm, and the cultures were incubated at about evening primroseA method for culturing a microorganism, which comprises diluting a fresh L B medium containing biotin to a cell density of approximately 0.05 OD600, incubating the mixture at 37 ℃ while shaking the mixture at 180rpm, mixing a donor and acceptor culture of 500. mu. L in a microtube of 1.5m L when the OD600 reaches 0.5 to 0.7, and allowing the mixture to stand at 37 ℃ for 2 minutes to 2 hours, preferably 5 minutes to 1 hour, more preferably 10 minutes to 30 minutes, and further preferably 15 minutes or so, wherein a step of conjugal transfer which conventionally requires several hours or more can achieve sufficient transfer in approximately 15 minutes according to the method of the present invention, continuously diluting the mixture, spreading the mixture on L B well plates containing antibiotics in various combinations to proliferate colonies and form colonies, measuring Colony Forming Units (CFU) on each of the well plates to calculate the transformation efficiency by conjugal transfer, and determining the CFU/total acceptor of the transformed CFU × 10/total acceptor⁶(ppm)。

[ Process (3) ]

In this step, the desired DNA fragment is accumulated in the chromosome of the recipient bacterium. Specifically, the desired DNA fragment can be accumulated in the chromosome of the recipient bacterium by repeating the steps (1) and (2) using the transformed cell obtained by the conjugative transfer in the step (2) as the recipient bacterium.

In the transformation method of the present invention, when the donor strain and the recipient strain are heterogeneous, the DNA to be transferred by ligation may be degraded in the recipient strain by a restriction enzyme or the like. In such a case, it is considered that in the step (1), the donor strain is transformed by introducing the methylase into the donor strain in advance in order to perform the same DNA modification (methylation modification, etc.) as in the recipient strain. For example, when a thermophilic bacterium (Geobacillus kaustophilus (GK)) is used as the recipient strain, the method disclosed in Japanese patent application laid-open No. 2011-211968 can be used. Specifically, the plasmid transfer donor to GK can further improve efficiency by performing DNA methylase addition and gene manipulation for forced enhancement of rap gene expression. In addition, the plasmid transferred to GK (pGK1) can be used: for example, pGR16B is added with ori for replication in GK, and a kanamycin resistance gene KmR (TK101) that is resistant to high temperatures required for selection in GK.