JPWO2019156152A1 - Nucleic acid containing arabinose-dependent gene expression control sequence - Google Patents

Abstract

The present disclosure describes a nucleic acid containing a gene control sequence capable of controlling the expression of a target gene in a coryneform bacterium, a bacterium into which the nucleic acid has been introduced, a method for expressing the target gene using the bacterium, and a substance using the bacterium. Regarding the production method. The nucleic acid has at least a portion of an arbitrary promoter sequence and an ArarR binding sequence identified by a predetermined nucleotide sequence. In addition, when the nucleic acid is introduced into a coryneform bacterium, the promoter activity of the promoter sequence is suppressed when arabinose is not added, and the promoter activity is induced when arabinose is added.

Description

The present invention relates to nucleic acid sequences that allow arabinose or analog-dependent gene expression control in coryneform bacteria. More specifically, the present invention relates to nucleic acids comprising a gene expression control sequence having a nucleotide sequence capable of functioning as a promoter in a coryneform bacterium and an AraR binding sequence consisting of a predetermined nucleotide sequence.

Since Corynebacterium glutamicum was isolated as a glutamic acid-producing bacterium in Japan in the 1950s, Corynebacterium-type bacteria represented by the same species have been chemicals containing various amino acids, organic acids, alcohols including ethanol, and the like. Widely used in the production of goods. In addition, basic research on coryneform bacteria such as genome analysis and gene expression analysis has been energetically carried out, and gene recombination technology for coryneform bacteria has been almost established. With the progress of basic research and the establishment of gene recombination technology in this way, the types of chemicals that can be produced by coryneform bacteria are increasing, and mass production of these chemicals is becoming possible. ..
As a result, the use of coryneform bacteria in the production of chemicals and useful substances is gaining increasing attention in the industry.

By the way, when a substance is produced using a coryneform bacterium into which various genes have been introduced, if the expression of various genes introduced into the coryneform bacterium can be artificially controlled, efficient bioprocess and substance production will be possible. Therefore, it is important to develop a gene expression control technique using a promoter or the like that can function in coryneform bacteria.

As an example of a promoter that functions in a coryneform bacterium, there is a technique using a promoter sequence of an aspartase coding gene isolated from the Brevibacterium flavum MJ-233 strain (Patent Document 1). In Patent Document 1, only the negative control plasmid in which the promoter sequence does not exist was introduced in the strain into which the reporter plasmid containing the predetermined promoter sequence and the chloramphenicol acetyltransferase (CAT) gene was introduced. It is described that 25 times the CAT activity was confirmed as compared with the strain.

As yet another example, it is known that some DNA fragments isolated from the genomic DNA of the Brevibacterium flavum MJ-233 strain exhibit a stronger promoter function than the tac promoter in Corynebacterium. (Patent Document 2). According to Patent Document 2, the promoter activity of these DNA fragments depends on the composition of the carbon source (various sugars, ethanol, proteolytic products, etc.) of the medium for culturing the host corine-type bacterium. It is described that the expression of the target gene can be controlled by changing the carbon source composition.

As yet another example, the promoter of _EF-TU ( _PEF-TU ), which is one of the elongation factor genes, the promoter of the superoxide dimethase gene (P _sod ), and the chaperonin protein derived from corynebacterium glutamicum. There are known expression units using the promoter of the GroES gene (P _gro ) and the promoter of _EF-TS (P _EF-TS ), which is one of the elongation factor genes (Patent Documents 3 to 6 respectively). In addition, a gene expression technique using the promoter sequences of the Cgl1565 gene (locus NCgl1504) and the Cgl1360 gene (locus NCgl1305) derived from the Corinebacterium glutamicum ATCC13032 strain is also known (Patent Document 7).
In Patent Documents 3 to 7, a plasmid construct obtained by introducing each of the above promoter sequences and a predetermined reporter gene or enzyme gene is constructed, and a corynebacterium glutamicum transformant into which the plasmid construct is introduced is obtained. However, it is described that the promoter activity was confirmed for each of the above promoter sequences by the reporter assay using the transformant or the measurement of the enzyme activity.

Further, Patent Document 8 describes an expression cassette or vector using a promoter sequence derived from the Brevibacterium ammoniagenes CJHB100 strain. These promoter sequences are promoter sequences identified by culturing the strain, identifying proteins overexpressed in each culture face, and cloning the 5'untranslated region of the gene encoding those proteins. In Patent Document 7, a total of 7 promoter sequences named pcj1 to pcj7 are obtained, and the promoter activity of these promoter sequences is evaluated by a reporter assay using the GFP gene in Brevibacterium ammoniagenes. As a result, it is suggested that the five promoter sequences except pcj3 and 4 show stronger promoter activity with respect to the typical promoter tac promoter, and in particular pcj1 and pcj4 show more than 10-fold promoter activity. Has been done. In addition, Patent Document 7 confirms that these promoter sequences can also function in Escherichia coli ( Escherichia coli ), and in particular, pcj1 may exhibit high activity in both coryneform bacteria and Escherichia coli. It has been suggested.

Further, although the promoter sequences disclosed in Patent Documents 1 to 8 are wild-type promoter sequences of coryneform bacteria, mutant promoter sequences in which the promoter activity is improved by introducing mutations into the wild-type promoter sequences are also available. Are known. Such mutant promoter sequences include, for example, the promoter sequences of the diaminopimelic acid dehydrogenase (ddh) gene, the LysC-asd operon gene, and the aspartate aminotransferase (aspB) gene derived from corynebacterium glutamicum. Examples thereof include mutant promoters obtained by introducing a predetermined mutation (Patent Documents 9 to 11). A series of techniques relating to promoters described in Patent Documents 9 to 11 aims to improve the production amount of lysine by increasing the expression level of an enzyme gene involved in the lysine biosynthesis pathway.

Further, Patent Document 12 contains two or more promoter sequences of _{PEF -TU} , P _sod , P _gro , and _{PEF -TS} derived from Corynebacterium glutamicum described in Patent Documents 3 to 6. Multiple promoters linked in tandem have been disclosed, and the results show that the expression of the target gene is improved in the multiple promoters as compared with the case where each of these promoters is used alone.

Further, as a technique using an inducible promoter, for example, a gene expression control technique disclosed in Patent Document 13 can be mentioned. The technique disclosed in Patent Document 13 utilizes a promoter sequence obtained by comprehensively analyzing the gene promoter of Corynebacterium glutamicum, which is induced or suppressed under anaerobic conditions. In Patent Document 13, if a promoter sequence capable of promoting or suppressing gene expression under anaerobic conditions is used in this way, various substances necessary for producing a target substance when producing a target substance under anaerobic conditions using a coryneform bacterium are used. It is suggested that more efficient production of the target substance is possible because the expression of the gene can be improved and the expression of various genes unnecessary for the production of the target substance can be suppressed. ..

Furthermore, expression systems that use the functions available heterologous promoter in Corynebacterium glutamicum has been known for a long time, is in such expression systems, e.g. _{tac, trc, LacUV5, P R} , P L , etc. Examples thereof include an Escherichia coli expression system using the above (see, for example, Non-Patent Documents 1 to 3). The expression of promoter activity of these promoters is induced by the addition of an inducer such as lactose or its analog isopropylthiogalactoside (IPTG).

In addition, a heat-induced expression vector that combines the λPL promoter with the latent promoters CJ1 and CJ4 isolated from Corynebacterium ammonia Genes is known, and this heat-induced expression vector is Corynebacterium ammonia. It has been confirmed that it functions not only in Genes but also in Corynebacterium glutamicum (Non-Patent Document 4).

In addition, the expression of the arabinose-dependent target gene in corynebacterium glutamicum by combining the AraC gene of the arabinose regulator derived from Escherichia coli, the P _BAD promoter of the araBAD operon and the AraE gene of the L-arabinose transporter. An expression system that enables control is also known (Non-Patent Document 5). Non-Patent Document 5 describes that the expression of the target gene could be strictly controlled without showing subbasal non-specific expression, and further, α-ketoglutarate dehydrogenation in coryneform bacteria using this expression system. It is stated that regulation of the expression of the odhI gene, which encodes an inhibitor of the enzyme, resulted in high levels of glutamate production.

Furthermore, in Non-Patent Document 6, in the Corinebacterium glutamicum ATCC31831 strain, the AraR protein, which is a LacI-type transcriptional regulator, has the araBDA gene responsible for L-arabinose catabolism, the araE gene responsible for arabinose absorption, and It has been suggested that the expression of the transcriptional regulator AraR gene is suppressed. More specifically, a specific sequence (BS _B ) in which the AraR protein is located between each gene region of araBDA and a gene region of GalM / araR, and two specific sequences (BS _E1 and BS) located upstream of the araE gene. It has been shown that it can bind to a total of three sequences of _E2 ), suggesting the role of the AraR protein and the AraR binding sequence in releasing the suppression of gene expression by L-arabinose. Non-Patent Document 6 also shows the results of evaluating the promoter regions of galM and araR by the LacZ reporter assay, and for the promoter region of AraR, cells cultured with D glucose added and L-arabinose were added. It is described that no difference in promoter activity was observed between the cells cultured in the above-mentioned cells, and that the expression level of LacZ increased in response to the addition of L-arabinose for the galM promoter.

JP-A-7-31478 JP-A-7-9591 Special table 2007-514425 Special table 2007-514426 Special table 2007-536908 Special table 2008-506400 Special table 2010-515453 Special table 2008-523802 Special table 2011-510651 Special table 2011-510625 Special table 2011-182779 Special table 2008-523834 WO2006 / 028063

Gene. 1991 Jun 15; 102 (1): 93-8. Appl Environ Microbiol. 1997 Nov; 63 (11): 4392-400. Nat. Biotechnol. 6: 428-430 J. Microbiol. Biotechnol. (2008), 18 (4), 18 (4), 639-647. Appl Environ Microbiol. 2012 Aug; 78 (16): 5831-5838. J Bacteriol. 2015 Dec; 197 (24): 3788-3996.

As described above, various gene expression systems that can be used in coryneform bacteria have been conventionally known. However, in the prior art, in which only constitutive promoters capable of functioning in coryneform bacteria are used alone, on / off switching control as a gene expression switch is practically impossible, for example, strictly. In the first place, it is not suitable for bioprocesses in which various gene regulation is indispensable.

In addition, regarding an expression system (for example, Non-Patent Documents 1 to 4) that controls the expression of a target gene by exposing a recombinant coryneform bacterium under a predetermined stress environment such as an expression inducer such as IPTG or temperature. There is a problem of toxicity and cost of the inducer, a problem that the expression control process of the target gene becomes complicated, and a certain level of non-specific expression may be observed even under the basal in which the expression is not induced, so that the gene is strict. There are also situations in which it is often difficult to control expression. In addition, depending on the nature of the target gene, there is also a problem that such non-specific expression under the basal inhibits the growth of coryneform bacteria.

Furthermore, regarding the gene expression system that combines the AraC gene, P _BAD promoter, and AraE gene derived from Escherichia coli, which is described in Non-Patent Document 5, the effect confirmation test by the present inventor using a coryne-type bacterium. When arabinose was added, about 10 times as much promoter activity was observed as compared with the case where arabinose was not added, but no remarkable promoter activity as described in Non-Patent Document 5 was observed. That is, the gene expression system described in Non-Patent Document 5 has poor reproducibility and cannot be said to be practically usable. In the first place, the gene expression system described in Non-Patent Document 5 utilizes a promoter sequence or gene derived from Escherichia coli as described above, and the gene expression system according to the present invention is a nucleotide sequence and an amino acid. The composition is completely different at the sequence level.

In addition, in Non-Patent Document 6, the AraR protein suppresses the expression of the araBAD gene, the araE gene and the transcriptional regulator AraR gene in the corynebacterium glutamicum ATCC31831, and the AraR protein is a gene of the arabinose gene group. Although it has been suggested that it can bind to a predetermined sequence in the interregional region, it does not describe the specific configuration of a gene expression control system using these gene groups and the AraR protein binding sequence at a feasible level. .. Actually, as shown in Comparative Test Example 1 of the following examples, when the present inventor confirmed the promoter region of the araE gene containing the AraR protein binding sequence, the gene was found even if the promoter region of the araE gene was used as it was. Expression control was impossible, and even promoter activity was not shown as a gene expression system in the first place. That is, Non-Patent Document 6 is an academic paper simply showing the analysis results on the role of the araR protein in the arabinose gene expression regulation mechanism of corynebacterium glutamicum, and is an academic paper showing the arabinose gene group and the AraR protein binding sequence in the artificial gene expression system. It does not imply availability.

Therefore, an object of the present invention is to provide a gene expression system capable of strictly controlling the expression of a target gene in a coryneform bacterium and ensuring a good expression level when inducing the expression of the target gene.

In order to achieve the above object, according to the first aspect of the present invention, the following nucleic acids are provided.
[1] A gene expression control sequence (R) having at least a part of a nucleotide sequence (X) capable of functioning as a promoter and the nucleotide sequence (Y) described in the following (a) or (b) is included.
Nucleic acid: The gene expression control sequence (R) satisfies the following condition (I).
(A) The nucleotide sequence according to any one of SEQ ID NOs: 5 to 12 and SEQ ID NOs: 17 and 18;
(B) A nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence according to (a) above.
However, when the nucleic acid is RNA, thymine (t) in the nucleotide sequence shall be read as uracil (u).
Condition (I): In a coryneform bacterium, the promoter activity of the nucleotide sequence (X) is suppressed when arabinose is not added.

[2] The nucleic acid according to [1], wherein the nucleotide sequence (Y) is directly or indirectly linked to the 3'end of the nucleotide sequence (X).
[3] The nucleic acid according to [1] or [2], wherein the nucleotide sequence (X) is a constitutive promoter.

[4] The nucleotide sequence (X) is trc promoter, tacI promoter, tacII promoter, T5 promoter, T7 promoter, lac promoter, trp promoter, tet promoter, EFTu promoter, groES promoter, SOD promoter, P15 promoter, ldhA promoter, gapA. The nucleic acid according to any one of [1] to [3], which is selected from a promoter, a dapA promoter, a metE promoter, and a tuf promoter.

[5] The nucleotide sequence (X) is infected with a promoter sequence derived from 5'upstream of a gene present in the genomic DNA of a coryneform bacterium, a promoter sequence derived from a plasmid inherent in the coryneform bacterium, or a coryneform bacterium. The nucleic acid according to any one of [1] to [4], which comprises a promoter sequence derived from the genome of the obtained phage.
[6] The gene expression control sequence (R) shows at least 10-fold higher promoter activity in coryneform bacteria when arabinose is added than in the case where arabinose is not added, from [1] to [1]. 5] The nucleic acid according to any one of.

[7] The gene expression control sequence (R) shows at least 15-fold higher promoter activity in coryneform bacteria when arabinose is added than in the case where arabinose is not added, from [1] to [1]. 6] The nucleic acid according to any one of.
[8] The gene expression control sequence (R) shows at least 20 times higher promoter activity in coryneform bacteria when arabinose is added than in the case where arabinose is not added, from [1] to [1]. 7] The nucleic acid according to any one of.

[9] The gene expression control sequence (R) shows at least 35 times higher promoter activity in coryneform bacteria when arabinose is added than when arabinose is not added, [1] et al. [ 8] The nucleic acid according to any one of.
[10] The gene expression control sequence (R) shows at least 50-fold higher promoter activity in coryneform bacteria when arabinose is added than in the case where arabinose is not added, from [1] to [1]. 9] The nucleic acid according to any one of.
[11] The gene expression control sequence (R) shows at least 70-fold higher promoter activity in coryneform bacteria when arabinose is added than when arabinose is not added, from [1] to [1]. 10] The nucleic acid according to any one of.

[12] The nucleic acid according to any one of [1] to [11], wherein the nucleotide sequence (Y) is represented by the following general formula (I):
5'-N ₁ TGTN ₂ AGCGN ₃ TN ₄ AN ₅ N ₆ N ₇ -3'... General formula (I)
In the formula, N ₁ , N ₂ , N ₃ , N ₄ , N ₅ , N ₆ and N ₇ independently indicate A (adenine), G (guanine), C (cytosine) or T (thymine), respectively. Or, if it is deleted and the nucleic acid is RNA, T (thymine) shall be read as U (uracil).

[13] The nucleic acid according to any one of [1] to [12], wherein the gene expression control sequence (R) contains any of the following nucleotide sequences (l) to (o):
(L) The nucleotide sequence according to any one of SEQ ID NOs: 22 to 61 and SEQ ID NOs: 96 to 102;
(M) A nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence according to (l);
(N) A nucleotide sequence that hybridizes with a nucleotide sequence complementary to the nucleotide sequence described in (l) under stringent conditions; and a sequence of at least 80% or more of the nucleotide sequence described in (o) (l). Nucleotide sequences with identity,
However, when the nucleic acid is RNA, thymine (t) in the base sequence should be read as uracil (u).

[14] The nucleic acid according to any one of [1] to [13], wherein the gene expression control sequence (R) further contains an SD sequence.
[15] The nucleic acid according to any one of [1] to [14], wherein the SD sequence is directly or indirectly linked to the 3'end of the nucleotide sequence (Y).
[16] The nucleic acid according to any one of [1] to [15], further comprising a nucleotide sequence encoding at least one of the araE protein and the araR protein.

[17] The nucleic acid according to any one of [1] to [16], which comprises a nucleotide sequence encoding at least one of an araE protein and an araR protein derived from a coryneform bacterium.
[18] One of [1] to [17] further comprising one or more nucleotide sequences selected from the group consisting of an origin of replication, a selectable marker gene, a cloning site, a restriction enzyme recognition site, and a terminator. The nucleic acid of the description.
[19] The nucleic acid according to any one of [1] to [18], which is provided as a plasmid capable of autonomous replication in bacteria.

[20] The nucleic acid according to any one of [1] to [19] provided as an expression vector.
[21] A nucleotide sequence (Z) encoding a target gene is further contained, and the nucleotide sequence (Z) controls gene expression so that the expression of the target gene is controlled by the gene expression control sequence (R) in coryneform bacteria. The nucleic acid according to any one of [1] to [18], which is directly or indirectly linked to the sequence (R).
[22] The nucleic acid according to any one of [1] to [21], which is DNA.

Furthermore, according to the second aspect of the present invention, the following bacteria are provided.
[23] A bacterium into which the nucleic acid according to any one of [1] to [22] has been introduced.
[24] The bacterium according to [23], which is a bacterium belonging to the genus Escherichia.
[25] The bacterium according to [23], which is a coryneform bacterium.

[26] A DNA having at least one nucleotide sequence encoding an araE protein and an araR protein derived from a coryneform bacterium is incorporated into the genomic DNA, and the genomic DNA can express the araE protein and the araR protein. The bacterium according to any one of [23] to [25], which is composed.

Furthermore, according to the third aspect of the present invention, the following method for expressing the target gene is provided.
[27] A method for expressing a target gene, which comprises expressing the target gene by exposing the bacterium according to any one of [23] to [26] to arabinose or an analog thereof.
[28] The method according to [27], wherein the bacterium is a coryneform bacterium.

Further, according to the fourth aspect of the present invention, the following method for producing the target substance is provided.
[29] A method for producing a target substance, which comprises expressing the target gene by exposing the bacterium according to any one of [23] to [26] to arabinose or an analog thereof.
[30] The method according to [29], wherein the bacterium is a coryneform bacterium.

Furthermore, according to the fifth aspect of the present invention, the following nucleic acid fragments are provided.
[31] A nucleic acid fragment for use in controlling the expression of a target gene in a coryneform bacterium.
Nucleic acid fragment containing the nucleotide sequence (Y) according to (a) or (b) below:
(A) The nucleotide sequence according to any one of SEQ ID NOs: 5 to 9, 11 and 12 and SEQ ID NOs: 17 and 18;
(B) A nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence according to (a) above.
However,
When the nucleic acid is RNA, thymine (t) in the nucleotide sequence shall be read as uracil (u).
It is assumed that the nucleotide sequences shown in SEQ ID NOs: 10, 20 and 21 as the nucleotide sequence (Y) are excluded, and the nucleotide sequence (Y) satisfies the following condition (II).
Condition (II): Corine-type bacterium transformed with a plasmid vector in which the nucleotide sequences from the 2913th to the 2928th positions in the nucleotide sequence of the plasmid vector pGE728-1 shown in SEQ ID NO: 80 are replaced with the above nucleotide sequence (Y). In, the promoter activity of PtacI is suppressed when arabinose is not added as opposed to when arabinose is added.

According to the present invention, efficient and strict gene expression control becomes possible, and an efficient bioprocess can be provided. In addition, according to the present invention, when the transformant is exposed to arabinose, the expression-suppressed state of the target gene is released and the expression of the target gene can be promoted, which facilitates operations and operations in the bioprocess. ..
Hereinafter, embodiments or modifications that can be further adopted in the aspects of the present invention will be illustrated below, and the advantages and effects of the present invention will also be described in detail.

It is a figure which shows typically the structure of the plasmid vector constructed in an Example. (A) shows the structure of the plasmid vector pGE728-1 constructed in Test Example 1, and (b) shows the structure of the plasmid vector pGE716 constructed in Test Example 3. It is a figure which shows typically the structure of the plasmid vector constructed in Test Example 3. (A) shows the structure of the plasmid vector pGE837, and (b) shows the structure of the plasmid vector pAra1. It is a figure which shows the result of the reporter assay performed in Test Example 3. In FIG. 3, "non-added" refers to the sample without arabinose, "added" refers to the sample with arabinose added, and "induction rate" indicates the promoter activity value / sample without arabinose shown by the sample with arabinose added. The ratio (fold) of the promoter activity value is shown. It is a figure which shows the result of the reporter assay performed in Test Example 3. In FIG. 4, “unadded” refers to a sample without arabinose added. It is a schematic diagram which shows the promoter region of the araE gene in the genomic DNA of Corynebacterium glutamicum ATCC31831 strain.

[Nucleic acid]
According to the first aspect of the present invention, the following nucleic acids are provided.
A gene expression control sequence (R) containing at least a part of the nucleotide sequence (X) capable of functioning as a promoter and the nucleotide sequence (Y) described in (a) or (b) below.
Nucleic acid: The gene expression control sequence (R) satisfies the following condition (I).
(A) The nucleotide sequence according to any one of SEQ ID NOs: 5 to 12 and SEQ ID NOs: 17 and 18;
(B) A nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence according to (a) above.
However, when the nucleic acid is RNA, thymine (t) in the nucleotide sequence shall be read as uracil (u).
Condition (I): In a coryneform bacterium, the promoter activity of the nucleotide sequence (X) is suppressed when arabinose is not added.

More specifically, the "nucleic acid" according to the present invention is a nucleic acid for use in controlling the expression of a target gene in bacteria including coryneform bacteria. The nucleic acid according to the present invention may be provided in any form of DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). Further, the nucleic acid according to the present invention may be in the form of a single strand or a double strand. The nucleic acid is specifically an isolated nucleic acid, cDNA or cRNA.
Considering that DNA is usually a form that can function as an expression regulator of a target gene in bacterial cells, and that DNA is chemically more stable than RNA, the nucleic acid of the present invention is DNA. It is preferable that it is provided in the form of. However, even when provided in the form of RNA, the gene expression control function prescribed in the present invention is exerted if RNA is converted to DNA in in vitro or in vivo, so that the form of DNA is used. It is not limited and may be provided in the form of RNA. Techniques for converting RNA to DNA using reverse transcriptase or the like are known to those skilled in the art. In addition, in the present invention, the nucleic acid may be chemically modified such as methylation.

In addition, the nucleic acid according to the present invention may be any nucleic acid as long as the gene expression control sequence (R) satisfies the above (I) when the promoter activity is measured by a reporter assay or the like as described later. The nucleic acid may or may not have a nucleotide sequence encoding the target gene. The nucleic acid according to the embodiment that does not contain the nucleotide sequence encoding the target gene is specifically an expression plasmid construct (nucleotide sequence encoding the target gene) to be tested for expression of the target gene in bacteria including coryneform bacteria. Is an expression cassette or expression vector (ie, before the gene of interest is inserted into a given cloning site) for use in the construction of (introduced).

In the present invention, the "coryneform bacterium" refers to a group of microorganisms as defined in Bergey's Manual of Determinative Bacteriophage, Vol. 8, p. 599, 1974.
More specifically, as coryneform bacteria, Corynebacterium spp., Brevibacterium spp., Arthrobacter spp., Mycobacterium spp., Micrococcus spp. ) Bacteria, Microbacterium spp. And the like.

Examples of Corynebacterium spp. Include the following species and strains.
Corynebacterium glutamicum (eg, FERM P-18976 strain, ATCC13032 strain, ATCC31831 strain, ATCC13058 strain, ATCC13059 strain, ATCC13060 strain, ATCC13232 strain, ATCC13286 strain, ATCC13287 strain, ATCC1375 strain, ATCC1373 strain , ATCC13761 strain, ATCC14020 strain);
Corynebacterium acetoglutamicum (eg, ATCC15806 strain);
Corynebacterium acetoacidophilum (eg, ATCC13870 strain);
Corynebacterium melassecola (eg, ATCC17965 strain);
Corynebacterium efficiens ;
Corynebacterium alkanoliticum (eg, ATCC21511 strain);
Corynebacterium carlunae (eg, ATCC15991 strain);
Corynebacterium lilium (eg, ATCC15990 strain);
Corynebacterium thermoaminogenes ( Corynebacterium efficiens ) (eg, AJ12340 strain, FERM BP1539 strain);
Corynebacterium herculis (eg, ATCC13868 strain).
Corynebacterium ammoniagenes ( Brevibacterium ammoniagenes ) (eg, ATCC6781 strain, ATCC6782 strain).

Specific examples of Brevibacterium spp. Examples include the following species and strains.
Brevibacterium divaricatum (Brevibacterium divaricatum) (for example ATCC14020 shares);
Brevibacterium flavum [for example, MJ-233 (FERM BP-1497) strain, MJ-233AB-41 (FERM BP-1498) strain, ATCC13826 strain, ATCC14067 strain, ATCC13826 strain];
Brevibacterium imariophyllum (eg, ATCC14068 strain);
Brevibacterium lactofermentum (Corynebacterium glutamicum) (Brevibacterium lactofermentum (Corynebacterium glutamicum) ) ( for example ATCC13869 shares);
Brevibacterium roseum (eg, ATCC13825 strain);
Brevibacterium saccharoliticum (eg, ATCC14066 strain);
Brevibacterium thiogenitalis (eg, ATCC19240 strain);
Brevibacterium album (eg, ATCC15111 strain);
Brevibacterium cerinum (eg, ATCC15112 strain).

Specific examples of the genus Earthrobacter include the following species and strains.
Examples thereof include Arthrobacter globiformis (for example, ATCC8010 strain, ATCC4336 strain, ATCC21056 strain, ATCC31250 strain, ATCC31738 strain, ATCC35698 strain) and the like.

Specific examples of Micrococcus spp. Are Micrococcus freudenreichi [for example, No. 239 (FERM P-13221) strain]; Micrococcus luteus [eg, No. 240 (FERM P-13222) strain]; Micrococcus ureae (for example, IAM1010 strain); Micrococcus roseus (for example, IFO3764 strain) and the like.
Specific examples of Microbacterium spp . Are Microbacteriaium ammoniaphilum (eg, ATCC15354 strain).

ATCC is an abbreviation for American Type Culture Collection (PO Box 1549 Manassas, VA 20108 USA), and the ATCC strain can be sold by the same institution. ..

Furthermore, in the present invention, the coryneform bacterium may be a wild-type strain originally existing in nature, or a set in which genomic DNA or plasmid DNA is manipulated or a predetermined gene, artificial sequence, or the like is introduced into these DNA. It may be a substitute. Furthermore, the coryneform bacterium may be a mutant strain created by exposure to a predetermined chemical substance or environmental conditions.

In the present invention, the "nucleotide sequence (X) capable of functioning as a promoter in a coryneform bacterium" refers to a nucleotide sequence exhibiting promoter activity in any one or more of the bacterial species / strains belonging to the coryneform bacterium as described above. Point to. The nucleotide sequence (X) may be an artificially synthesized sequence or a natural sequence present in an organism containing bacteria.

In the present invention, the term "promoter" is used in a sense generally understood by those skilled in the art. More specifically, the "nucleotide sequence (X) capable of functioning as a promoter in coryneform bacteria" refers to RNA polymerase that specifically binds to coryneform bacteria and initiates transcription into mRNA by the transcriptional activity of the RNA polymerase. Means a nucleotide sequence that can be allowed.
In the art, in addition to a nucleotide sequence capable of purely initiating transcription into mRNA by the transcriptional activity of RNA polymerase, an operator sequence (for example, lacO), a ribosome binding sequence (Shine-Dalgarno sequence; SD sequence), etc. Although the term "promoter" may be used to include the gene control sequence of RNA in the present invention, in principle, the term "promoter" refers only to a nucleotide sequence capable of initiating transcription into mRNA purely by the transcriptional activity of RNA polymerase. However, in the nucleic acid according to the present invention, the existence of the additional gene control sequence as described above is not excluded.

As described above, the nucleotide sequence (X) is not particularly limited as long as it can function as a promoter in any one or more of the bacterial species / strains belonging to the coryneform bacterium, but it is convenient. In consideration, the embodiment in which a nucleotide sequence exhibiting promoter activity is commonly used for all strains belonging to the coryneform bacterium is preferable. Furthermore, in some embodiments, as the nucleotide sequence (X), a nucleotide sequence capable of functioning as a promoter in the genus Escherichia (eg, Escherichia coli) in addition to the coryneform bacterium can be adopted.

Further, in some embodiments, the nucleotide sequence (X) is a promoter sequence derived from the 5'upstream region of a gene endogenous to the genomic DNA of a bacterium containing a coryneform bacterium as described above, autonomous replication in these bacteria. It contains a promoter sequence derived from a possible plasmid, a promoter sequence derived from the genome of a phage that infects these bacteria, and the like. In addition, in certain embodiments, the promoter sequences derived from these bacteria or phages may be wild-type sequences or may be wild-type sequences introduced with a predetermined mutation.
In general, the promoter sequence is located 5'upstream of the transcription initiation site and is known to be characterized by -35 box and -10 box. Therefore, primer extension method, quantitative RT-PCR, etc. It is also possible to determine the transcription start point by the known method of the above and obtain it in consideration of the information of the determined transcription start point and the like.
That is, in the present invention, a known promoter sequence may be used as the nucleotide sequence (X), but the present invention is not particularly limited to this, and the newly obtained promoter sequence as described above may be used or artificially used. You may use a newly created promoter.

Furthermore, as the type of promoter sequence that can be used as the nucleotide sequence (X) in the present invention, an inducible promoter capable of inducing the expression of a target gene by adding a specific culture condition or a chemical substance, or RNA polymerase. Examples include constitutive promoters that depend on availability and allow constant expression.

In the present invention, the type of promoter used as the nucleotide sequence (X) is not particularly limited, but a constitutive promoter is preferable. This is because, when the nucleotide sequence (X) can function as a constitutive promoter in particular, the presence of the nucleotide sequence (Y) prescribed in the present invention causes the nucleotide sequence (X) under the basal basis when arabinose is not added. ) Strictly suppresses the promoter activity, and the suppressed state of the promoter activity can be released by a simple operation of adding arabinose, and significant expression of the target gene can be rapidly induced.

More specifically, the nucleotide sequence (X) may be a promoter of an enzyme gene involved in various amino acid biosynthesis systems in bacteria including coryneform bacteria or a derivative thereof (mutation-introduced sequence). More specifically, glutamic acid biosynthetic enzyme gene (for example, glutamate dehydrogenase gene), glutamine biosynthetic enzyme gene (for example, glutamine synthase gene), lysine biosynthetic enzyme gene (for example, aspart kinase gene), threonine bio Synthetic genes (eg, homoserine dehydrogenase genes), isoleucine and valine biosynthetic enzyme genes (eg, acethydroxyic acid synthase genes), leucine biosynthetic enzyme genes (eg, 2-isopropylapple acid synthase genes), proline and Aromatic amino acid biosynthetic enzyme genes such as arginine biosynthetic enzyme genes (eg glutamate kinase gene), histidine biosynthetic enzyme genes (eg phosphoribosyl-ATP pyrophosphorylase gene), tryptophan, tyrosine and phenylalanine (eg deoxyarabinohepts) Nucleic acid biosynthetic enzyme genes such as rhonic acid phosphate (DAHP) synthase gene), inosic acid and guanylate (eg, phosphoribosyl pyrophosphate (PRPP) amide transferase gene, inosic acid dehydrogenase gene and guanylate synthesis Examples thereof include each promoter such as (enzyme gene) or a derivative thereof (mutation-introduced sequence).

Further, the nucleotide sequence (X) may be a promoter of a gene encoding a factor involved in cell division, and examples thereof include a promoter of the divIVA gene.
In addition, promoters that operate during the logarithmic growth phase as nucleotide sequence (X) are also preferably used, for example, diviVA, gap, ldhA, fda, glyA, cysK, aroF, gpmA, eno, humC, pfk, sdhA, mdh, argF, Promoters of various genes such as proA, proC, aceE, serA, metE, nifS1, tpi, aceD, cysD, sdhB, and pack can be mentioned.

Further, as the nucleotide sequence (X), a strong promoter (strong promoter) used in the Escherichia coli expression system such as trc promoter, tac promoter, T5 promoter, T7 promoter, lac promoter, trp promoter and tet promoter is also preferably used. To.
In addition, the nucleotide sequence (X) is disclosed in EF-Ts promoter, EF-Tu promoter, groES promoter, SOD promoter, P15 promoter, gapA promoter, dapA promoter, tuf promoter, metE promoter, and Patent Documents 1 to 13. Various promoters can also be used.

Next, the nucleotide sequence (Y) will be described. The gene expression control sequence (R) in the nucleic acid according to the present invention includes the nucleotide sequence (Y) described in (a) or (b) above in addition to the nucleotide sequence (X).

The nucleotide sequence shown in (a) is the nucleotide sequence set forth in any one of SEQ ID NOs: 5 to 12. These nucleotide sequences are derived from the AraR protein binding sequence present on the genomic DNA of Corynebacterium glutamicum. Their nucleotide sequences are shown below.
(1) 5'-ATGTTAGCGCTAACAT-3'(SEQ ID NO: 5)
(2) 5'-ATGTGAGCGATAACAC-3'(SEQ ID NO: 6)
(3) 5'-ATGTGAGCGCTAACAC-3'(SEQ ID NO: 7)
(4) 5'-ATGTGAGCGCTAACTC-3'(SEQ ID NO: 8)
(5) 5'-ATGTGAGCGTTAAAAT-3'(SEQ ID NO: 9)
(6) 5'-ATGTGAGCGCTAAACT-3'(SEQ ID NO: 10) (BS _E1 )
(7) 5'-ATGTTAGCGCTGACAT-3'(SEQ ID NO: 11)
(8) 5'-ATGTGAGGCGCTGACAT-3'(SEQ ID NO: 12)
(9) 5'-GTGTGAGCCGTACAC-3'(SEQ ID NO: 17)
(10) 5'-GTGTGAGCGATACAC-3'(SEQ ID NO: 18)

As shown in the following examples, a nucleic acid (expression vector) using the nucleotide sequence set forth in any one of SEQ ID NOs: 5 to 12 and SEQ ID NOs: 17 and 18 as the nucleotide sequence (Y) was introduced into a coryneform bacterium. In this case, the promoter activity can be strictly suppressed under the basal to which arabinose is not added, and the promoter activity can be significantly induced by releasing the suppressed state by adding arabinose. In order to reliably suppress the promoter activity in the situation where arabinose is not added and to secure the gene expression control effect capable of inducing the promoter activity at a high level by the addition of arabinose, the nucleotide sequences of (a) and (b) It is preferable to use the nucleotide sequences specified in (i) to (vi) below as a reference.
(I) The nucleotide sequence according to any one of SEQ ID NOs: 5 to 8 and SEQ ID NOs: 10, 11 and 17;
(Ii) More preferably, the nucleotide sequence according to any one of SEQ ID NOs: 5 to 8, 10 and 17;
(Iii) Even more preferably, the nucleotide sequence according to any one of SEQ ID NOs: 6 to 8, 10 and 17;
(Iv) Even more preferably, the nucleotide sequence set forth in any one of SEQ ID NOs: 6, 7, 10 and 17;
(V) Even more preferably, the nucleotide sequence set forth in any one of SEQ ID NOs: 6, 7 and 17;
(Vi) Most preferably the nucleotide sequence set forth in SEQ ID NO: 17.
Furthermore, in order to realize the above-mentioned gene expression control effect at a more remarkable level, it is even more preferable to adopt the nucleotide sequence defined in the above-mentioned (a) as the nucleotide sequence (Y), and among those nucleotide sequences, It is even more preferable to adopt the nucleotide sequence shown in any of the above (i) to (vi), and a higher effect can be expected from (i) to (vi).

In the present invention, as the nucleotide sequence (Y), it is defined in (b) above on the premise that the gene expression control sequence (R) satisfies the condition (I) in addition to the nucleotide sequence defined in (a) above. The nucleotide sequence to be used can also be adopted.
That is, the nucleotide sequence defined in (b) is the nucleotide sequence set forth in any one of SEQ ID NOs: 5 to 12 and SEQ ID NOs: 17 and 18, in which one or more bases are deleted, substituted or added. It is a nucleotide sequence.

Here, the range of "one or more" is not particularly limited as long as the gene expression control sequence (R) satisfies the condition (I). The range includes, for example, 1 to 10, 1 to 9, 1 to 8, preferably 1 to 7, more preferably 1 to 6, still more preferably 1 to 5, and particularly preferably 1 to 1. It can be 4, 1 to 3, 1 to 2, or 1.

Further, in a specific embodiment, the nucleotide sequence (Y) is a nucleotide sequence represented by the general formula (I) shown below.
5'-N ₁ TGTN ₂ AGCGN ₃ TN ₄ AN ₅ N ₆ N ₇ -3'... General formula (I)

In the formula, N ₁ , N ₂ , N ₃ , N ₄ , N ₅ , N ₆ and N ₇ independently represent A (adenine), G (guanine), C (cytosine) or T (thymine), respectively. .. Further, N ₁ is preferably A (adenine) or G (guanine), N ₂ is preferably G (guanine) or T (thymine), and N ₃ is A (adenine), C. (Cytocin) or T (thymine) is preferred, N ₄ is preferably A (adenine), C (citosine) or G (guanine), and N ₅ is A (adenine) or C (citosine). ), N ₆ is preferably A (adenine), C (cytosine) or T (thymine), and N ₇ is preferably C (citosine) or T (thymine). However, when the nucleic acid is RNA, T (thymine) shall be read as U (uracil).

Additionally, in certain embodiments, the general formula any location (e.g., location 1 to 3 or 1 to 2 positions) of the nucleotides in the N ₇ from N ₁ in the nucleotide sequence represented by the formula (I) have been deleted The nucleotide sequence can be adopted as the nucleotide sequence (Y). Further, in a more specific embodiment, the nucleotide sequence in which any one or both of N ₂ and N ₃ is deleted in the nucleotide sequence represented by the general formula (I) is the nucleotide sequence (Y). May be adopted as.

In the nucleic acid of the present invention, the positional relationship between the nucleotide sequence (X) and the nucleotide sequence (Y) is not particularly limited as long as the gene expression control sequence (R) satisfies the condition (I).
For example, the nucleotide sequence (Y) may be directly or indirectly linked to the 5'end of the nucleotide sequence (X), or the nucleotide sequence (Y) may be directly or indirectly linked to the 3'end of the nucleotide sequence (X). May be done. In this case, the nucleotide sequence (Y) may be present in an untranslated region or an intergenic region, or may be present in a region encoding a predetermined gene (open reading frame).
In addition, embodiments in which at least a portion of the nucleotide sequence (X) and at least a portion of the nucleotide sequence (Y) overlap may also be included in the present invention. Specific examples of the embodiment in which at least a part of the nucleotide sequence (X) and at least a part of the nucleotide sequence (Y) overlap are as follows.
i) A form in which the entire nucleotide sequence (Y) is contained within the nucleotide sequence (X) region;
ii) A form in which a part of the 3'side of the nucleotide sequence (Y) overlaps with a part of the 5'side of the nucleotide sequence (X);
iii) A form in which a part of the 5'side of the nucleotide sequence (Y) overlaps with a part of the 3'side of the nucleotide sequence (X).

As described above, the positional relationship between the nucleotide sequences (X) and (Y) is not particularly limited, but in order to reliably produce the above-mentioned predetermined effect, the nucleotide sequence (Y) is the nucleotide sequence (X). ) Is preferably directly or indirectly connected to the 3'end.
Here, "the nucleotide sequence (Y) is indirectly linked to the nucleotide sequence (X)", "the 3'end or 5'end of the nucleotide sequence (Y) is the 5'end or 3'of the nucleotide (Y). A phrase such as "indirectly linked to the end" means that the nucleotide sequence (X) and the nucleotide sequence (Y) are linked via a sequence consisting of one nucleotide or a plurality of nucleotides. The number of nucleotides in the "sequence consisting of a plurality of nucleotides" is not particularly limited as long as the gene expression control sequence (R) satisfies the condition (I), but is, for example, 2 to 50, preferably 2 to 40. , More preferably 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, and even more preferably 2 to 9, 2 to 8 and 2 to. 7 pieces, 2 to 6 pieces, 2 to 5 pieces, 2 to 4 pieces, 2 to 3 pieces. In addition, the "sequence consisting of a plurality of nucleotides" connecting the nucleotide sequence (X) and the nucleotide sequence (Y) may be a simple linker sequence having no specific function, or a further promoter sequence. , Ribosome binding sequence (Shine-Dalgarno sequence; SD sequence), other transcriptional regulatory sequence, translational regulatory sequence, or other sequence having a gene expression regulatory function.

On the other hand, "the nucleotide sequence (Y) is directly linked to the nucleotide sequence (X)", "the 3'end or 5'end of the nucleotide sequence (Y) is the 5'end or 3'end of the nucleotide (Y). Phrases such as "directly linked to" may be understood literally, and the nucleotide sequence (X) and the nucleotide sequence (Y) do not go through one or more other nucleotides as described above. It means that it is directly connected to.

Further, each of the nucleotide sequence (X) and the nucleotide sequence (Y) may be present one by one or a plurality of each in the nucleic acid of the present invention. In addition, when a plurality of nucleotide sequences (X) or nucleotide sequences (Y) are present, the plurality of nucleotide sequences (X) or nucleotide sequences (Y) have exactly the same sequences as each other. It may have a different sequence.

Next, the condition (I) will be described.
In the present invention, the significance of the gene expression control sequence (R) satisfying the condition (I) is as follows.

As described above, in the nucleic acid according to the present invention, the gene expression control sequence (R) is a nucleotide sequence (X) that functions as a promoter that controls the expression of the target gene, and an AraR binding sequence that is present in the genomic DNA of coryneform bacteria. The nucleotide sequence derived from is included as the nucleotide sequence (Y).

Due to the composition of the nucleotide sequences (X) and (Y), the AraR protein binds to the nucleotide sequence (Y) in the coryneform bacterium into which the nucleic acid prescribed in the present invention has been introduced in an environment where the arabinose concentration is below the basal level. As a result, it is presumed that the promoter activity of the nucleotide sequence (X) is suppressed. However, on the other hand, when the coryneform bacterium is exposed to an environment in which the arabinose concentration exceeds the basal level, the AraR protein undergoes allosteric regulation by arabinose and dissociates from the nucleotide sequence (Y), whereby the nucleotide sequence ( It is presumed that the promoter activity of X) is induced and the expression of the target gene is promoted. That is, as a phenomenon actually observed when the expression of the target gene is controlled by using the nucleic acid according to the present invention, the promoter activity by the nucleotide sequence (X) is suppressed in an environment where the arabinose concentration is below the basal level. Expression of a given gene, which is in the state of being controlled by the promoter activity of nucleotide sequence (X), is suppressed at the level of transcription into mRNA or protein expression. On the other hand, in an environment where the arabinose concentration exceeds the basal level, the expression of the predetermined gene is promoted at the level of mRNA transcription or protein expression.
Therefore, the gene expression control sequence (R) is a condition (I) that the promoter activity of the nucleotide sequence (X) is suppressed when arabinose is not added to the coryneform bacterium when arabinose is added. It is necessary to satisfy.
Here, the sufficiency of condition (I) can be confirmed by the reporter assay shown below.

That is, a DNA fragment obtained by linking a nucleotide sequence encoding a reporter gene to the 3'end of the gene expression control sequence (R) constructed as described above is introduced into a predetermined site of an expression vector capable of functioning in coryneform bacteria. Prepare the DNA construct. Here, examples of the reporter gene include β-galactosidase gene (LacZ), β-glucuronidase, chloramphenicol acetyltransferase, various fluorescent proteins (for example, green fluorescent protein) and the like. More specifically, a nucleotide sequence encoding a reporter gene is directly or indirectly linked to the 3'end of the gene expression control sequence (R), such as an expression vector plasmid having the nucleotide sequence shown in SEQ ID NO: 80 or 81. It is sufficient to prepare a DNA construct, that is, a reporter plasmid in which the DNA fragment is inserted. Such reporter plasmids may be constructed independently or may utilize various commercially available reporter plasmid systems for reporter assays.
Then, a transformant obtained by introducing such a DNA construct into a coryneform bacterium is obtained.
The transformant is cultured for a certain period of time using a medium containing arabinose at a predetermined concentration or higher and a medium containing arabinose at a concentration lower than the predetermined concentration, and the culture medium is sampled. For each of the bacterial cells derived from the sampled culture medium, the mRNA of the reporter gene is quantified by a method such as quantitative PCR, or the activity of the reporter protein expressed from the reporter gene is appropriately measured. Satisfiability of condition (I) can be determined by determining how many times the mRNA expression level or reporter protein activity value measured for the former culture medium sample is multiple of that measured for the latter culture medium sample. it can.

More specifically, in the present invention, the sufficiency of condition (I) can be confirmed by, for example, the reporter assay method shown in the following Examples.
That is, in the plasmid vector pGE728-1 (SEQ ID NO: 80) used in Test Example 1 of the following examples, the promoter sequence (PtacI) and the AraR binding sequence existing upstream of the β-galactosidase gene (LacZ), which is a reporter gene, By exchanging the region (SEQ ID NO: 32) in which the two are linked with the gene control sequence (R) of interest for which the sufficiency of condition (I) is evaluated, and performing a reporter assay using the obtained plasmid vector. It is convenient to confirm the sufficiency of the condition (I) of the gene control sequence (R). This is because a method for evaluating promoter activity using a β-galactosidase gene (LacZ) has already been established as a well-known and commonly used assay system by those skilled in the art, so that experimental operations are easy and data reliability and reproducibility are good.
In the reporter assay, the assay conditions including the induction conditions by adding arabinose, the medium composition, the culture conditions such as the culture temperature and the culture time, etc. may be particularly limited as long as a reliable promoter activity ratio can be obtained. However, the assay conditions may be appropriately adjusted in consideration of the properties of the gene expression control sequence (R) to be selected, the type and properties of the reporter gene to be used, the properties of the coryneform bacterium to be used, and the like.

More specifically, the β-galactosidase gene (LacZ) assay is described, for example, in J. et al. Biol. Chem. , 1995, 270: 11181-11189 or Genetics, 2010, 185: 823-830. The specific procedure is shown below as an example.

(1) The transformant obtained by introducing the above DNA construct into the Corynebacterium glutamicum ATCC13032 strain was inoculated into a predetermined amount of liquid medium (4% glucose, 25 μg / mL kanamycin) and at a predetermined temperature. OD ₆₁₀ = 0.5 to 1.5 (optionally _OD 610 = 0.5 to 1) to cultured to. When OD ₆₁₀ = 0.5 to 1.5 (in some cases, OD ₆₁₀ = 0.5 to 1), a predetermined amount of culture solution was transferred to another culture vessel, and arabinose was added to a predetermined final concentration. Prepare the one that has been prepared and the one that does not contain arabinose.
(2) Each of the above cultures was cultured for a predetermined time, the cultures with and without arabinose induction were sampled, OD (610 nm) was measured, and 20 μL was 80 μL of permeation buffer (100 mM Na ₂ HPO ₄ , 20 mM KCl, Mix with 2 mM sulfonyl ₄ , 0.8 mg / mL CTAB, 0.4 mg / mL sodium deoxycholate, 5.4 μL / mL β-mercaptoethanol).
(3) Mix with 600 μL of substrate solution (60 mM Na ₂ HPO ₄ , 40 mM NaH ₂ PO ₄ , 1 mg / mL ONPG, 2.7 μL / mL β-mercaptoethanol) and incubate at 25 ° C.
(4) Add 700 μL of 1 M Na ₂ CO ₃ to stop the reaction, and centrifuge at 15,000 g for 5 minutes.
(5) The absorbance (A420) of the supernatant is measured.
(6) Based on the following formula 2, the mirror unit (Miller Unit) is calculated as the promoter activity.
1 mirror unit = 1000 × (A420 / t × V × OD610) ・ ・ ・ Equation (II)
In the formula, t indicates the time (minutes) and V indicates the culture solution (mL).

As the liquid medium, for example, a medium having the composition shown below can be used.
Yeast extract 2g, cazaminoic acid 1g, urea 2g, sulfuric acid 7g, dipotassium hydrogen phosphate 0.5g, potassium dihydrogen phosphate 0.5g, magnesium sulfate heptahydrate 0.5g, iron sulfate heptahydrate 6 mg, manganese sulfate monohydrate 4.2 mg, biotin 0.2 mg, thiamine 0.2 mg (per 1 L).

As described above, by comparing the promoter activity value measured for the culture solution to which arabinose was added and the promoter activity value measured for the culture solution to which arabinose was not added, arabinose was compared with the promoter activity observed when arabinose was added. The gene expression control sequence (R), which can be said to have significantly suppressed promoter activity observed when not added, satisfies the condition (I), and is a nucleotide sequence (Y) which is a component of the nucleic acid according to the present invention. ) Can be adopted.

Here, in the gene expression control sequence (R), the promoter activity value measured for a sample in which the promoter activity of the nucleotide sequence (X) is induced by the addition of arabinose or the like is measured for a sample to which arabinose is not added as a control. It is preferably at least 5 times higher than the promoter activity, more preferably at least 10 times higher, and even more preferably at least 15 times, 20 times, 25 times, 30 times, 35 times, 40 times or 45 times. It is high, particularly preferably at least 50 times, 55 times, 60 times, 65 times, 70 times, 80 times, 90 times, 100 times or 150 times higher.
In addition, here, the meaning of "inducing promoter activity by adding arabinose or the like" means only arabinose in the embodiment in which the promoter activity of the nucleotide sequence (X) is controlled only by the nucleotide sequence (Y). However, when another gene expression regulator (for example, lacO) is used in addition to the nucleotide sequence (Y), it is regulated by the other gene expression regulator in addition to the addition of arabinose. It means that the necessary operations are required.
In addition, the gene expression control sequence (R) is, paradoxically, a promoter whose promoter activity value measured for a control sample without arabinose is measured for a sample in which the promoter activity of nucleotide sequence (X) is induced. It is preferably 1/5 or less of the activity value, and in this case, more preferably 1/10 or less, still more preferably 1/15 or less, 1/20 or less, 1/25 or less, 1/30 or less, 1 /. 35 or less, 1/40 or less or 1/45 or less, particularly preferably 1/50 or less, 1/55 or less, 1/60 or less, 1/65 or less, 1/70 or less, 1/80 or less, 1/90 or less , 1/100 or less, or 1/150 or less.

Furthermore, although it depends on the type of reporter assay selected, measurement conditions, etc., when the reporter assay conditions shown in the above procedures (1) to (6) or Examples are adopted, arabinose-free sample is used. The promoter activity measured is preferably 10 mirror units or less. Further, the upper limit of the promoter activity measured for the sample to which arabinose is not added is preferably 9, more preferably 8, 7, 6, 5, 4, particularly preferably 3.5, 3.4, 3.3. 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2 It is 0.0, 1.9, 1.8, 1.7, 1.6, 1.2, 1.1, 1.0 (unit is mirror unit).

As a preferred embodiment, a gene expression control sequence (R) containing any of the following nucleotide sequences (p) to (s) can be adopted.
(P) The nucleotide sequence according to any one of SEQ ID NOs: 22 to 41;
(Q) A nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence according to (p);
(R) A nucleotide sequence that hybridizes with a nucleotide sequence complementary to the nucleotide sequence described in (p) under stringent conditions; and a sequence of at least 80% or more of the nucleotide sequence described in (s) (p). Nucleotide sequences with identity,
However, when the nucleic acid is RNA, thymine (t) in the nucleotide sequence should be read as uracil (u).

Each nucleotide sequence shown in SEQ ID NOs: 22 to 41 is shown below.
(1-1): 5'-TTGACAATTAATCATCATCGGCTCGTAATAATGTGGGA ATGTTAGCGCTAAACT- 3'(SEQ ID NO: 22)
(1-2): 5'-TTGACAATTAATCATCGGCTCGTAATAATGTTGGA ATGTGAGCGATAACAC -3'(SEQ ID NO: 23)
(1-3): 5'-TTGACAATTAATCATCGGCTCGTAATAATGTTGGA ATGTGAGCGCTAACAC- 3'(SEQ ID NO: 24)
(1-4): 5'-TTGACAATTAATCATCGGCTCGTAATAATGTTGGA ATGTGAGCGCTAACTC- 3'(SEQ ID NO: 25)
(1-5): 5'-TTGACAATTAATCATCGGCTCGTAATAATGTTGGA ATGTGAGCGTTAAAAT -3'(SEQ ID NO: 26)
(1-6): 5'-TTGACAATTAATCATCGGCTCGTAATAATGTTGGA ATGTGAGCGCTAAACT -3'(SEQ ID NO: 27)
(1-7): 5'-TTGACAATTAATCATCGGCTCGTAATAATGTGGGA ATGTTAGCGCTGACAT -3'(SEQ ID NO: 28)
(1-8): 5'-TTGACAATTAATCATCGGCTCGTAATAATTGTGGA ATGTGAGGCGCTGACAT -3'(SEQ ID NO: 29)
(1-9): 5'-TTGACAATTAATCATCATCGGCTCGTAATAATGTGTGGGA GTGTGAGGCTAAACCA- 3'(SEQ ID NO: 30)
(1-10): 5'-TTGACAATTAATCATCGGCTCGTAATAATGTGTGGGA GTGTGAGCGATACAC -3'(SEQ ID NO: 31)

(1-11): 5'-GAGCTGTTGACAATTAATCATCGGCTCGTAATAAGTGTGGA ATGTTAGCGCTAACAT -3'(SEQ ID NO: 32)
(1-12): 5'-GAGCTGTTGACAATTAATCATCGGCTCGTAATAAGTGTGGA ATGTGAGCGATAACAC -3'(SEQ ID NO: 33)
(1-13): 5'-GAGCTGTTGACAATTAATCATCGGCTCGTAATAAGTGTGGA ATGTGAGCGCTAACAC -3'(SEQ ID NO: 34)
(1-14): 5'-GAGCTGTTGACAATTAATCATCGGCTCGTAATAAGTGTGGA ATGTGAGCGCTAACTC -3'(SEQ ID NO: 35)
(1-15): 5'-GAGCTGTTGACAATTAATCATCGGCTCGTAATAAGTGTGGA ATGTGAGCGTTAAAAT -3'(SEQ ID NO: 36)
(1-16): 5'-GAGCTGTTGACAATTAATCATCGGCTCGTAATAATGTGTGGA ATGTGAGCGCTAAACT -3'(SEQ ID NO: 37)
(1-17): 5'-GAGCTGTTGACAATTAATCATCGGCTCGTAATAAGTGTGGA ATGTTAGCGCTGACAT -3'(SEQ ID NO: 38)
(1-18): 5'-GAGCTGTTGACAATTAATCATCGGCTCGTAATAAGTGTGGA ATGTGAGGCGCTGACAT -3'(SEQ ID NO: 39)
(1-19): 5'-GAGCTGTTGACAATTAATCATCGGCTCGTAATAAGTGTGGA GTGTGAGCGCTAACAC -3'(SEQ ID NO: 40)
(1-20): 5'-GAGCTGTTGACAATTAATCATCGGCTCGTAATAATGTGTGGGA GTGTGAGCGATACAC- 3'(SEQ ID NO: 41)

Furthermore, as another preferred embodiment, a gene expression control sequence (R) containing any of the following nucleotide sequences (t) to (s) can be adopted.
(T) The nucleotide sequence according to any one of SEQ ID NOs: 42 to 61;
(U) A nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence according to (t);
(V) A nucleotide sequence that hybridizes with a nucleotide sequence complementary to the nucleotide sequence of (t) under stringent conditions; and a sequence of at least 80% or more of the nucleotide sequence of (w) (t). Nucleotide sequences with identity,
However, when the nucleic acid is RNA, thymine (t) in the nucleotide sequence should be read as uracil (u).

The nucleotide sequences shown in SEQ ID NOs: 42 to 61 are shown below.
(2-1): 5'-TTGACAATTAATCATCGAACTAGTTTAATGTTGGA ATGTTAGCGCTAAACT- 3'(SEQ ID NO: 42)
(2-2): 5'-TTGACAATTAATCATCGAACTAGTTTAATGTTGGA ATGTGAGCGATAACAC -3'(SEQ ID NO: 43)
(2-3): 5'-TTGACAATTAATCATCGAACTAGTTTAATGTGTGGA ATGTGAGCGCTAACAC- 3'(SEQ ID NO: 44)
(2-4): 5'-TTGACAATTAATCATCGAACTAGTTTAATGTTGGA ATGTGAGCGCTAACTC -3'(SEQ ID NO: 45)
(2-5): 5'-TTGACAATTAATCATCGAACTAGTTTAATGTGTGGGA ATGTGAGCGTTAAAAT -3'(SEQ ID NO: 46)
(2-6): 5'-TTGACAATTAATCATCGAACTAGTTTAATGTGTGGGA ATGTGAGCGCTAAACT -3'(SEQ ID NO: 47)
(2-7): 5'-TTGACAATTAATCATCGAACTAGTTTAATGTTGGA ATGTTAGCGCTGACAT -3'(SEQ ID NO: 48)
(2-8): 5'-TTGACAATTAATCATCGAACTAGTTTAATGTTGGA ATGTGAGCGCTGACAT -3'(SEQ ID NO: 49)
(2-9): 5'-TTGACAATTAATCATCGAACTAGTTTAATGTGTTGGA GTGTGAGCGCTAACAC -3'(SEQ ID NO: 50)
(2-10): 5'-TTGACAATTAATCATCGAACTAGTTTAATGTGTTGGA GTGTGAGCGATACAC -3'(SEQ ID NO: 51)

(2-11): 5'-GAGCTGTTGACAATTAATCATCGAACTAGTTTAATGTGTGGA ATGTTAGCGCTACATA- 3'(SEQ ID NO: 52)
(2-12): 5'-GAGCTGTTGACAATTAATCATCGAACTAGTTTAAGTGTGGA ATGTGAGCGATAACAC -3'(SEQ ID NO: 53)
(2-13): 5'-GAGCTGTTGACAATTAATCATCGAACTAGTTTAATGTGTGGGA ATGTGAGCGCTAACAC -3'(SEQ ID NO: 54)
(2-14): 5'-GAGCTGTTGACAATTAATCATCGAACTAGTTTAATGTGTGGGA ATGTGAGCGCTAACTC -3'(SEQ ID NO: 55)
(2-15): 5'-GAGCTGTTGACAATTAATCATCGAACTAGTTTAATGTGTGGGA ATGTGAGCGTTAAAAT -3'(SEQ ID NO: 56)
(2-16): 5'-GAGCTGTTGACAATTAATCATCGAACTAGTTTAATGTGTGGGA ATGTGAGCGCTAAACT -3'(SEQ ID NO: 57)
(2-17): 5'-GAGCTGTTGACAATTAATCATCGAACTAGTTTAATGTGTGGA ATGTTAGCGCTGACAT -3'(SEQ ID NO: 58)
(2-18): 5'-GAGCTGTTGACAATTAATCATCGAACTAGTTTAATGTGTGGGA ATGTGAGCGCTGACAT -3'(SEQ ID NO: 59)
(2-19): 5'-GAGCTGTTGACAATTAATCATCGAACTAGTTTAATGTGTGGGA GTGTGAGCGCTAACAC -3'(SEQ ID NO: 60)
(2-20): 5'-GAGCTGTTGACAATTAATCATCGAACTAGTTTAATGTGTGGGA GTGTGAGCGATACAC- 3'(SEQ ID NO: 61)

The underlined sequence in the sequences (1-1) to (1-20) and (2-1) to (2-20) corresponds to the nucleotide sequence (Y), and the other 5'upstream region is the nucleotide sequence. Corresponds to (X).
The nucleotide sequences (Y) in the gene expression control sequences (R) shown in (1-1) to (1-20) correspond to the nucleotide sequences shown in SEQ ID NOs: 5 to 12 and SEQ ID NOs: 17 and 18, respectively. The nucleotide sequences (Y) in the gene expression control sequences (R) shown in (2-1) to (2-20) also correspond to the nucleotide sequences shown in SEQ ID NOs: 5 to 12 and SEQ ID NOs: 17 and 18, respectively.

On the other hand, the nucleotide sequence (X) in the sequences shown in (1-1) to (1-10) is the −35 box, -10 box and transcription of PtacI frequently used in the expression system in Escherichia coli (E. coli). It is a core sequence containing the starting site (+1) and is considered to be necessary and sufficient for the expression of promoter activity (Li et al. Microbial Cell Factories 2012, 11:19). On the other hand, the nucleotide sequence (X) in the gene control sequence (R) shown in (1-11) to (1-20) is a transcription initiation site (+1) in the PtacI sequence generally recognized by those skilled in the art. ) Corresponds to the sequence including the entire area upstream from), and 6 nucleotides are further added to the 5'end of the core sequence (Proc. Natl. Acad. Sci. USA, Vol. 80, pp. 21). -25, January 1983).

When PtacI (SEQ ID NO: 1 or 2) is used as the nucleotide sequence (X), it is possible to control the expression of the target gene particularly efficiently as shown in the examples. Therefore, from (p) to (s) above. It is preferable to use the indicated nucleotide sequence as the gene expression control sequence (R).

However, it is also preferable to use PtacII (SEQ ID NO: 3 or 4) as the nucleotide sequence (X) as well as PtacI, and in this case, more specifically, the nucleotide sequences shown in (t) to (w) above are included. It is preferable to adopt the gene expression control sequence (R). The nucleotide sequence (X) in the sequences shown in (2-1) to (2-10) defined in (t) above is the -35 box of PtacII often used in the expression system in Escherichia coli (Escherichia coli). , -10 box and transcription initiation site (+1), which is considered necessary and sufficient for the expression of promoter activity (see the above document). Furthermore, the nucleotide sequence (X) in the sequences shown in (2-11) to (2-20) is a sequence containing the entire area upstream from the transcription initiation site (+1) in the PtacII sequence generally recognized by those skilled in the art. This is because (6 nucleotides are further added to the 5'end of the core sequence), and high promoter activity can be expressed as in PtacI (see the above document).

Furthermore, as another preferred embodiment, a gene expression control sequence (R) containing any of the following nucleotide sequences (h) to (k) can be adopted.
(H) The nucleotide sequence according to any one of SEQ ID NOs: 96 to 102;
(I) A nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence according to (h);
(J) A nucleotide sequence that hybridizes with a nucleotide sequence complementary to the nucleotide sequence of (h) under stringent conditions; and a sequence of at least 80% or more of the nucleotide sequence of (k) (h). Nucleotide sequences with identity,
However, when the nucleic acid is RNA, thymine (t) in the nucleotide sequence should be read as uracil (u).

Each of the nucleotide sequences shown in SEQ ID NOs: 96 to 102, as the nucleotide sequence (X), in order, is a tuf promoter, a dapA promoter, a metE promoter, an ldhA promoter, a gapA promoter, and a sod, which are present on the genome of the corynebacterium glutamicum ATCC13031 strain. The promoter and the nucleotide sequence of the tuf promoter are included, and the nucleotide sequence shown in SEQ ID NO: 5 as the nucleotide sequence (Y) is included. The nucleotide sequence of SEQ ID NO: 102 is obtained by changing the linking site of the AraR-binding sequence to the tuf promoter with respect to the nucleotide sequence of SEQ ID NO: 96, which also employs the tuf promoter. The putative transcription region 5'terminal region is overlapped with the 5'terminal region of the AraR binding sequence.

Furthermore, as yet another embodiment, for the embodiment specified by (h) to (k) above, the nucleotide sequence in the nucleotide sequence according to any one of SEQ ID NOs: 96 to 102 according to (h). An embodiment including various modifications in which the region of X) (that is, the nucleotide sequence portion according to SEQ ID NO: 5) is replaced with the nucleotide sequence set forth in any one of SEQ ID NOs: 6 to 12 and SEQ ID NOs: 17 and 18. The form (V) can be mentioned.

In the nucleotide sequences shown in (q), (u) and (i) in each of the above-described embodiments and other nucleotide sequences corresponding thereto, the range of "one or more" is conditioned on the gene expression control sequence (R). As long as (I) is satisfied, it is not particularly limited. The range is, for example, 1 to 40, 1 to 35, 1 to 30, 1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1 to 12 1 to 10, preferably 1 to 9, 1 to 8, 1 to 7, more preferably 1 to 6, even more preferably 1 to 5, particularly preferably 1 to 4, 1 to It can be three, one to two, or one.

Further, with respect to the nucleotide sequences shown in (r), (v) and (j) in each of the above-described embodiments and the other nucleotide sequences in the present invention, the term "nucleotide sequence that hybridizes under stringent conditions" is specifically defined. Specifically, in a nucleic acid hybridization method such as a colony hybridization method, a plaque hybridization method, or a Southern blot hybridization method, the nucleotide sequence described in (p), (t), or (h) is used as a reference. It means a nucleotide sequence that forms a complex complementary to a nucleotide sequence that is complementary to the nucleotide sequence. Here, as stringent hybridization conditions, 6M urea, 0.4% SDS, 0.5 × SSC, or 0.1% SDS (60 ° C., 0.3 mol NaCl, 0.03 M sodium citrate) ), Or a stringency hybridization condition equivalent to these. Under conditions of higher stringency, such as 6M urea, 0.4% SDS, 0.1 × SSC, more homologous nucleotide sequences can be identified.

Furthermore, the nucleotide sequences shown in (s), (w) and (k) are nucleotide sequences having at least about 80% or more sequence identity with the nucleotide sequences described in (p), (t) and (h), respectively. Is. Further, the nucleotide sequences shown in (s), (w) and (k) have at least about 85% or more sequence identity with respect to the nucleotide sequences described in (p), (t) and (h), respectively. It is preferably a nucleotide sequence having, more preferably about 90% or more, and further preferably about 91% or more, 92% or more, 93% or more, 94% or more, 95% or more. , 96% or more, 97% or more, 98% or more, 99% or more sequence identity.
It should be noted that these matters can be similarly applied to the embodiment (V) according to the above-described modification, and the specific modification to which these matters are applied to the embodiment (V) is also described in the present specification. It is clearly disclosed in the book.

In addition, the gene expression control sequence (R) in the nucleic acid according to the present invention may further contain other gene expression regulatory sequences in addition to the nucleotide sequences (X) and (Y). Examples of such other gene expression regulatory sequences include further transcriptional regulatory sequences such as lac operators, translational regulatory sequences such as ribosome-binding sequences (Shine-Dalgarno sequence; SD sequence), and the like.
In the embodiment in which the nucleic acid according to the present invention is provided in the form of an expression cassette, an expression vector, or the like, for example, the SD sequences shown in (3-1) to (3-6) below may be contained.
(3-1) 5'-AGGA-3'
(3-2) 5'-GGT-3'
(3-3) 5'-GAAGGATT-3'
(3-4) 5'-GAAAGGAGG-3'
(3-5) 5'-GAAGGCGA-3'
(3-6) 5'-GAAGGAGA-3'

The position of the SD sequence is not particularly limited as long as the desired effect of the present invention can be obtained. From the viewpoint of more surely causing the expression control effect of the target gene by the nucleotide sequences (X) and (Y), the SD sequence exists between the 3'end of the nucleotide sequence (Y) and the start codon. In this case, the SD sequence is directly or indirectly linked to the 3'end of the nucleotide sequence (Y).

In addition, in the gene control sequence (R) according to the present invention, it is particularly preferable to adopt the SD sequence shown in (3-1). The SD sequence shown in (3-1) is an SD sequence associated with PtacI and PtacII, and as shown in the following examples, it ensures strict gene expression control of the target gene using arabinose. Moreover, a high gene expression level can be promised at the protein expression level.
More specifically, the embodiments according to (p) to (s), the embodiments (t) to (w), the embodiments (h) to (k), and the modifications thereof. In the embodiment (V), the nucleotide sequences specified in (p), (t) and (h) are directly linked to the 3'end of the nucleotide sequence shown in (4-1) below. Examples thereof include embodiments by reading each of the nucleotide sequences.
(4-1) 5'-TCACACAGGA-3' (SEQ ID NO: 62)

Furthermore, in embodiments where the nucleic acids according to the invention are provided, especially in the form of expression cassettes or vectors, the nucleic acids can function in bacteria (particularly Escherichia coli and / or coryneform bacteria) terminators (transcription termination). Signals) (eg, T _rrnB , T _GroEL , T _trp , T _T7 ) may be further included. The position of the terminator is not particularly limited as long as the desired effect of the present invention can be obtained, but in general, the terminator can be directly or indirectly linked to the 3'end of the nucleotide sequence (Y) or SD sequence. More specifically, when a cloning site is provided at the 3'end of the nucleotide sequence (Y) or SD sequence, the terminator can be directly or indirectly linked to the 3'end of the cloning site. Furthermore, in the embodiment in which the target gene coding region is linked to the 3'end of the nucleotide sequence (Y) or SD sequence, the terminator can be directly or indirectly linked to the 3'end of the target gene coding region.

Further, the nucleic acid according to the present invention may optionally further contain a nucleotide sequence encoding at least one of the araE protein and the araR protein. This is because if the araE protein and the araR protein are forcibly expressed in the cells, the certainty and strictness of gene expression control by arabinose addition can be ensured.
Considering that the nucleotide sequence (Y) is derived from the AraR binding sequence present in the genomic DNA of Corynebacterium glutamicum as described above, it is a coryneform bacterium, preferably a Corynebacterium spp., More preferably a corynebacterium. It is preferable that at least one of the araE gene coding sequence and the araR gene coding sequence present in the genomic DNA of Corynebacterium glutamicum is a nucleic acid (DNA) inserted.

Furthermore, the nucleic acid according to the present invention may further contain one or more nucleotide sequences selected from the group consisting of at least one origin of replication, a selectable marker gene, a cloning site and a restriction enzyme recognition site. The origin of replication and the selectable marker gene may be functional in all bacteria or in multiple species of bacteria, or may be functional in a particular bacterial species. For example, those containing any one or more of the following can be mentioned.
(1) Origin of replication and / or selectable marker gene coding sequence that can function only in coryneform bacteria;
(2) Origin of replication and / or selectable marker gene coding sequence that can function only in Escherichia (eg, Escherichia coli); and (3) Origin of replication that can function in both Corine-type bacteria and Escherichia. Selectable marker gene coding sequence.

Furthermore, the nucleic acid according to the present invention can be provided as a plasmid capable of autonomous replication in bacteria including Coryneform bacteria and Escherichia spp. In addition, the nucleic acids according to the invention can be provided as expression vectors.
The nucleic acid according to the present invention is provided, for example, in the form of a shuttle expression vector capable of autonomous replication in both Coryneform bacteria and Escherichia spp., And further capable of expressing a target gene in Coryneform bacteria. There may be.

Furthermore, the nucleic acid according to the present invention may further contain a nucleotide sequence (Z) that is directly or indirectly linked to the 3'end of the nucleotide sequence (Y) and encodes a gene of interest. However, as described above, the nucleotide sequence (Z) is not an essential component in the nucleic acid according to the present invention.

Furthermore, according to the fifth aspect of the present invention, the following nucleic acids are also provided.
A nucleic acid fragment for use in controlling the expression of a target gene in a coryneform bacterium.
Nucleic acid fragment containing the nucleotide sequence (Y) according to (a) or (b) below:
(A) The nucleotide sequence according to any one of SEQ ID NOs: 5 to 9, 11 and 12 and SEQ ID NOs: 17 and 18;
(B) A nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence according to (a) above.
However,
When the nucleic acid is RNA, thymine (t) in the nucleotide sequence shall be read as uracil (u).
It is assumed that the nucleotide sequences shown in SEQ ID NOs: 10, 20 and 21 as the nucleotide sequence (Y) are excluded, and the nucleotide sequence (Y) satisfies the following condition (II).
Condition (II): Corine-type bacterium transformed with a plasmid vector in which the nucleotide sequences from the 2913th to the 2928th positions in the nucleotide sequence of the plasmid vector pGE728-1 shown in SEQ ID NO: 80 are replaced with the above nucleotide sequence (Y). In, the promoter activity of PtacI is suppressed when arabinose is not added as opposed to when arabinose is added.
Here, the promoter activity of PtacI is preferably suppressed to 1/8 or less, more preferably 1/9 or less, 1/10 or less, 1/15 or less, 1/20 or less, 1/25 or less, and more preferably. Preferably 1/30 or less, 1/35 or less, 1/40 or less, 1/45 or less, particularly preferably 1/50 or less, 1/55 or less, 1/60 or less, 1/65 or less, 1/70 or less, It is 1/80 or less, 1/90 or less, 1/100 or less, or 1/150 or less.
By the way, the nucleotide sequences shown in SEQ ID NOs: 10, 20 and 21 are natural AraR binding sequences (BS _E1 , respectively) existing in the intergenic region of the arabinose gene group present on the genomic DNA of the Corinebacterium glutamicum ATCC31831 strain. It corresponds to BS _E2 , BS _B ) (Non-Patent Document 6).

The terms defining the nucleic acid according to the fifth aspect are as described for the nucleic acid according to the first aspect, and the above-described embodiments or features shown for the nucleic acid according to the first aspect are particularly inconsistent. As long as it does not occur, it can be adopted in the nucleic acid according to the fifth aspect, and various combinations of these embodiments or features are disclosed in the present specification as an embodiment that can be adopted in the nucleic acid according to the fifth aspect. Is.
Furthermore, the nucleic acid according to the fifth aspect can also be utilized in the further aspects shown below.

[Bacteria into which nucleic acid has been introduced]
Further, according to the second aspect of the present invention, a bacterium into which the nucleic acid according to the present invention has been introduced is provided.
The type of bacteria into which the nucleic acid according to the present invention has been introduced is not particularly limited, and examples thereof include Escherichia spp. And the above-mentioned Corynebacterium spp. (Preferably Corynebacterium spp.).
The bacterium into which the nucleic acid according to the present invention has been introduced is one in which a nucleic acid containing at least one nucleotide sequence encoding an araE protein and an araR protein derived from a coryneform bacterium is incorporated into genomic DNA so that these proteins can be expressed. You may. In this case, the nucleic acid according to the present invention introduced into the bacterium specifically has a nucleotide sequence (Z) encoding a target gene 3'downstream of the gene expression control sequence (R) (nucleotide sequence (Y)). It can be concatenated. This is because when the araE protein and the araR protein are forcibly expressed in the cells, the expression control of the target gene by the above-mentioned addition of arabinose is more reliably and efficiently realized. As described above, in the embodiment in which at least one nucleotide sequence encoding the araE protein and the araR protein is incorporated into the host genomic DNA, the nucleic acid according to the present invention similarly encodes the araE protein and the araR protein, respectively. It may not contain at least one nucleotide sequence, or may contain at least one nucleotide sequence.

[Method of expressing the target gene and method of producing the target substance]
According to a third aspect of the present invention, there is provided a method for expressing a target gene, which comprises expressing the target gene by exposing the bacterium into which the nucleic acid of the present invention has been introduced to arabinose or an analog thereof.
Furthermore, according to a fourth aspect of the present invention, there is provided a method for producing a target substance, which comprises expressing the target gene by exposing the bacterium into which the nucleic acid of the present invention has been introduced to arabinose or an analog thereof. To.
Hereinafter, the methods according to the third and fourth aspects of the present invention may be collectively referred to as the methods of the present invention.

The specific embodiment of "exposing the bacterium to arabinose or an analog thereof" in the method of the present invention is not particularly limited as long as the promoter activity is induced, but for example, a step of culturing the bacterium of the present invention. The bacterium may be exposed to arabinose by adding arabinose to the medium.
In this case, the concentration of arabinose or an analog thereof in the medium is not particularly limited as long as the promoter activity is induced, but is, for example, 0.001% or more, preferably 0.005% or more, more preferably 0. It is 007%, more preferably 0.008% or more, 0.009% or more, and particularly preferably 0.01% or more, 0.015% or more, 0.018% or more. In addition, the upper limit of the concentration of arabinose or its analog is not particularly limited as long as the promoter activity is induced, but in consideration of cost, it is 5% or less, preferably 3% or less, more preferably 2.5. % Or less. An arbitrary combination of these upper and lower limits is disclosed herein as a range of concentrations of arabinose or an analog thereof upon induction of promoter activity, and in certain embodiments of the invention. Can be adopted.
In the present invention, arabinose is more specifically L-arabinose.
Furthermore, in the present invention, the analog of arabinose (“the analog”) is the same gene expression control as arabinose in the presence of the gene control sequence (R) (nucleotide sequence (Y)) in the nucleic acid according to the present invention. A compound that exhibits the behavior of arabinose and has a basic skeleton of arabinose, but some atoms or atomic groups in the chemical structure of arabinose are replaced with atoms or atomic groups different from those of arabinose.

Further, the induction time of the promoter activity by the addition of arabinose (for example, the culture time in the presence of arabinose) may be appropriately adjusted so as to obtain the desired expression of the promoter activity, and is not particularly limited, but for example, 0. It can be 5 to 48 hours, preferably 1 to 24 hours, more preferably 1 to 12 hours, 1 to 6 hours. In a specific embodiment, as shown in the following examples, a sufficient increase in promoter activity and an expression level of a target gene can be secured in an extremely short time, so that the biotechnology is excellent in efficiency / productivity and economy. The process can be realized.

By the way, methods for expressing various genes and producing various substances using transformants of bacteria such as Coryneform bacteria and Escherichia spp. Are known, for example, from Patent Documents 1 to 13 and Non-Patent Documents 1 described above. 7 also describes various processes.
If the present invention is applied to these known processes, more efficient production of substances is possible, and the nucleic acids, transformants, and processes thereof obtained by applying the present invention in those known processes are also the present invention. Included in. However, the present invention is not limited to the case where the present invention is applied to a known process, and it goes without saying that the entire process to which the present invention is applied is included in the method according to the present invention.
From the examples of the processes shown in the examples of the present specification, it is clear that the nucleic acid according to the present invention can be applied to a method for expressing various genes and a method for producing a target substance.
In particular, in the method for producing the target substance according to the fourth aspect, the protein itself, which is the translation product of the target gene, may be produced as the target substance, or the protein, which is the translation product of the target gene, is intracellular. As a result of being produced in, a substance (for example, a metabolite) produced as a by-product may be produced as a target substance. In the latter case, examples of the protein that is the translation product of the target gene include an enzyme, and the recombinant enzyme expressed in the cells can promote metabolism and produce the target substance.

The nucleic acid of the present invention having the above-mentioned constitution is, for example, genomic DNA or plasmid DNA possessed by various bacteria including Corine-type bacteria and Escherichia spp., Genome nucleic acid possessed by various bacterial products capable of infecting these bacteria, artificially designed. It can be produced based on genetic engineering technology / molecular biological method including various cloning technology and mutation introduction technology by using a resource such as a nucleic acid fragment having a nucleotide sequence as a material. For genetic engineering techniques / molecular biology techniques including various cloning techniques and mutation introduction techniques, for example, Molecular Cloning: A Laboratory Manual, Fourth Edition (3-Volume Set) Cold Spring Harbor Laboratory Pr, etc. can be referred to. Those skilled in the art can produce the nucleic acid according to the present invention by using such a known technique with reference to the disclosure of the present specification. In addition, the nucleic acid according to the present invention may be partially or wholly produced by chemical synthesis. Bacteria according to the present invention can also be produced by referring to the contents disclosed in the present specification and various genetic engineering techniques / molecular biological techniques. An example of the nucleic acid / bacterium according to the present invention, a method for producing the same, and a method according to the present invention is shown in the following examples.

Although the specific embodiments of the present invention have been described in detail above, it goes without saying that the present invention is not limited to the above-described embodiments. Various modifications, modifications, and combinations may be adopted for each configuration, element, and feature without departing from the gist of the present invention.
Unless otherwise specified, the terms "including" and "having" in the present invention do not exclude the existence of elements other than the elements referred to as objects by these terms, and these terms are mixed. Will be done.

[Test Example 1]
In Test Example 1, an expression vector into which the araE gene and the araR gene, and the gene control sequence (R) and the target gene (reporter gene) according to the present invention were introduced was introduced into a coryneform bacterium, and the target gene (reporter gene) was introduced. An example in which the expression of is controlled is shown.
The details of the test procedure are shown below.

1. 1. Preparation of various expression vectors Various coryneform bacterium / E. coli shuttle vectors were prepared by the following procedure.

(1) PGEK003 and (KanR / pUCori / pCG1ori) Construction pHSG298 (Takara Bio) a template by PCR method A DNA fragment containing the kanamycin resistance gene (K a nR) and for E. coli origin of replication pUCori (high copy number), respectively Amplified.
Furthermore, a DNA fragment containing the origin of replication for Corynebacterium pCG1ori was amplified by the PCR method using the plasmid pCG1 carried by the Corynebacterium glutamicum ATCC13058 (NBRC12169) strain as a template.
K a nR, shown in SEQ ID NO: 63, 64 and 65 the nucleotide sequence of the amplified region by PCR for pUCori and PCG1ori.
In addition, pCG1 is extracted from the above strain according to a conventional method.

Then, using the In-Fusion HD Cloning Kit (Takara Bio), and the above DNA fragment by ligating ring in the order of K a nR / pUC ori / pCG1 ori, to prepare a plasmid vector PGEK003.

(2) Construction of pGEK008 (PldhA-rrrnBter / KanR / pUCori / pCG1ori) pGEK003 was cleaved with BamHI and EcoRV to obtain a linear DNA fragment. Note that this linear DNA fragment is one which is cleaved by the recognition site for the restriction enzyme in the region between the pCG1ori and K a nR.
Next, using the In-Fusion HD Cloning Kit, the PldhA fragment and the rrnBter fragment separately amplified by the PCR method were ligated in a circular manner to the pGEK003 cleavage fragment to obtain a plasmid vector pGEK008.
In the pGEK008, PldhA-rrnBter / K a nR / pUCori / pCG1ori are arranged in this order.
In addition, the Corynebacterium glutamicum ATCC13032 strain genomic DNA was used as a template for the amplification of the PldhA fragment, and the plasmid vector pFLAG-CTC (sigma) was used as a template for the amplification of the rrnBter fragment. The nucleotide sequences of these amplified fragments are shown in SEQ ID NOs: 66 and 67, respectively.

(3) Construction of pGEK020 (PldhA-lacZ-rrnBter / KanR / pUCori / pCG1ori) pGEK008 was cleaved with NdeI and BamHI to obtain a linear DNA fragment. The linear DNA fragment was cleaved at the recognition site of the restriction enzyme in the region between PldhA and rrnBter.
Furthermore, the LacZ gene fragment (LacZ fragment) was separately amplified by the PCR method using the template described later. At this time, NdeI site and BamHI site were inserted into the primer pair as adapter sequences, respectively, and the amplified LacZ fragment was similarly cleaved with NdeI and BamHI. Next, using the In-Fusion HD Cloning Kit, the pGEK008 cleavage fragment and the LacZ fragment were ligated in a circular manner to obtain a plasmid vector pGEK020.
In this pGEK020, PldhA-lacZ-rrnBter / K a nR / pUCori / pCG1ori are arranged in this order.
In addition, the plasmid vector owned by the applicant was used as a template for the amplification of the LacZ gene fragment. The LacZ gene region amplified in this applicant-owned plasmid vector is derived from pSV-β-Galactosidase Control Vector (Promega) and contains the same nucleotide sequence as the LacZ gene coding region of the vector.
When used as a LacZ gene as a reporter gene or a target gene, a commercially available plasmid containing the Escherichia coli genomic DNA or the lacZ gene coding region (for example, the above-mentioned plasmid vector) may be obtained and the LacZ gene coding region may be cloned.
The nucleotide sequence of the amplified LacZ gene fragment is shown in SEQ ID NO: 68.

(4) Construction of pGEK030 (MCS-lacZ-rrnBter / KanR / pUCori / pCG1ori) pGEK020 was cleaved with NdeI and NotI to obtain a linear DNA fragment from which the region from pCG1ori to PldhA was removed.
Amplified by PCR using a multi-cloning site (MCS; AgeI-SpeI-KpnI-NheI-XhoI-NdeI) constructed by annealing partially complementary synthetic oligonucleotides (SEQ ID NOs: 69 and 70) and pGEK020 as templates. The pCG1ori fragment and the linear DNA fragment were ligated in a circular manner using a Gibson Assembly Cloning Kit (New England Biolabs Japan) to obtain a plasmid vector pGEK030.
In this pGEK030, MCS-lacZ-rrnBter / K a nR / pUCori / pCG1ori are arranged in this order.
The nucleotide sequence of the amplified pCG1ori fragment is shown in SEQ ID NO: 71.

(5) Construction of pGEK094 (Ptac-lacZ-rrnBter / KanR / pUCori / pCG1ori) For the construction of pGEK094, another plasmid (hereinafter referred to as a derivative plasmid) obtained by introducing another element into the MCS of pGEK020 was used as a material. However, the difference between the derived plasmid and GEK020 is simply whether or not another element is inserted in the MCS, and the other element is deleted by treating the derived plasmid with KpnI and NdeI as follows when preparing pGEK094. It is a thing. As a result, the pGEK094 constructed in the present procedure (5) is equivalent to the one in which the Ptac fragment described below is inserted between the KpnI site and the NdeI site in the MCS of pGEK020. Therefore, the details of the step of preparing the derived plasmid actually used for the construction of pGEK094 are omitted.
Further, the Ptac fragment was obtained by amplifying the plasmid vector owned by the applicant by the PCR method as a template, and the Ptac region in the plasmid vector is derived from the commercially available plasmid vector pFLAG-CTC (sigma). It is a thing. Therefore, the Ptac fragment can be amplified using pFLAG-CTC (sigma) as a template. The nucleotide sequence of the actually amplified Ptac fragment is shown in SEQ ID NO: 72.
Then, the linear DNA fragment obtained by cleaving the above-mentioned derivative plasmid with KpnI and NdeI and the amplified Ptac fragment were ligated in a circular manner using a Gibson Assembly Cloning Kit to obtain pGEK094.

(6) Construction of pGEK122 (rrnBT1ter-T7ter-T7Uter-lacI / Ptac-lacZ-PldhA / KanR / pUCori / pCG1ori) Escherichia coli (DH5α) genomic DNA was used as a template to amplify the lacI gene fragment by the PCR method. In addition, using a plasmid vector (owned by the applicant) carrying the T7 terminator sequence as a template, the T7 terminator fragment (T7Uter-T7ter-rrnBT1ter) (ACS Synth Biol. 2015 Mar 20; 4 (3): pp265) was used by the PCR method. -273) was amplified. The nucleotide sequence of the amplified T7 terminator fragment is shown in SEQ ID NO: 73. In the nucleotide sequence of the T7 terminator used in this test example, the elements of T7Uter-T7ter-rrnBT1ter are linked in this order, and the order of the elements is different from that disclosed in the above document. In addition, the linker sequences present between the elements are also different from those disclosed in the above literature.
The lacI gene was inserted in the process of preparing various vectors by the inventor, but is finally removed in the vector according to the present invention as described below. Therefore, the information on the nucleotide sequence of the amplified lacI gene coding region is omitted.

Next, the pGEK094 was cleaved with KpnI and AgeI, and the obtained digested product was ligated with the amplified lacI gene fragment and the T7 terminator fragment by a Gibson Assembly Cloning Kit to obtain a plasmid vector pGEK122.

(7) Construction of pGE651 (PdapA-araE-rrnBT1ter-T7ter-T7Uter-lacI / Ptac-lacZ-rnBter / KanR / pUCori / pCG1ori) Corynebacterium glutamicum ATCC13032 strain Genomic DNA was used as a template, and pA fragment was used as a template. Amplified. In addition, the araE fragment was amplified by the PCR method using the genomic DNA of Corynebacterium glutamicum ATCC31831 strain as a template. The nucleotide sequences of the amplified PdapA region and araE gene coat region are shown in SEQ ID NOs: 74 and 75, respectively.

Further, pGEK122 was cleaved with the restriction enzyme XbaI, and the obtained digested product and the above PdapA fragment and araE fragment were ligated in a cyclic manner by In-Fusion HD Cloning Kit. The vector thus prepared was named pGE651.
(8) Construction of pGE677 (PdapA-araE-rrnBT1ter-T7ter-T7Uter-lacI / Ptac-lacZ-rrnBter / KanR / pSC101 / pCG1ori) Also, by processing the restriction enzymes NcoI and BamHI, the pGE651 pUC1 owner A vector was prepared by exchanging with the Escherichia coli origin of replication pSC101 ori (low copy number) contained in the plasmid vector of the above, and this was named pGE677.
The pSC101ori region in the above-mentioned applicant-owned plasmid vector is derived from the plasmid vector pMW119 (purchased from Takara Bio, currently available from Nippon Gene), and contains the same nucleotide sequence as the pSC101ori region of the vector.
The nucleotide sequence of the pSC101ori region portion containing the restriction enzymes NcoI and BamHI at both ends is shown in SEQ ID NO: 76.

(9) Construction of pGE689 (PdapA-araE-rrnBT1ter-T7ter / PdapAm-araR / Ptac-AraRBS-lacZ-rrnBter / KanR / pSC101 / pCG1ori) Corynebacterium glutamicum ATCC13032 strain APCd The DNA fragment containing the region was amplified. The nucleotide sequence of the amplified PdapAm fragment is shown in SEQ ID NO: 77. In addition, a mutation that increases the promoter activity by about twice is introduced into this PdapAm.
Furthermore, the araR gene fragment was amplified by the PCR method using the genomic DNA of the Corynebacterium glutamicum ATCC31831 strain as a template. The nucleotide sequence of the amplified araR gene coding region is shown in SEQ ID NO: 78.
Furthermore, using pGEK094 as a template, a fragment in which the ArarR-binding sequence was linked to Ptac was amplified by the PCR method. The nucleotide sequence of this fragment is shown in SEQ ID NO: 79.
By cleaving the pGE677 obtained in (8) above with restriction enzymes AgeI and NdeI, a linear DNA fragment from which the T7Uter and LacI regions have been removed is obtained, and this DNA fragment, as well as the above PdapA fragment, araR gene fragment and Ptac Fragments in which the araR-binding sequence was linked to were ligated in a cyclic manner by a Gibson Assembly Cloning Kit to prepare pGE728-1.
The structure of pGE728-1 is shown in FIG. 1 (a), and its nucleotide sequence is shown in SEQ ID NO: 80.

(10) Mutagenesis into the AraR binding sequence in pGE728-1
Design several primer pairs of degenerate primers or transformation-introduced primers that cover the AraR binding sequence site, amplify the entire plasmid using pGE728-1 as a template in the same manner as PCR, and then cut the template DNA with the restriction enzyme DpnI. Then, by transforming into Escherichia coli, a plasmid vector in which mutations were introduced semi-randomly into the AraR binding sequence was obtained. Specifically, Escherichia coli transformant colonies were randomly selected, and the introduction of mutations was confirmed by sequencing the nucleotide sequences of the AraR-binding region of the plasmid vectors carried by these transformants.
In this way, plasmid vectors pGE728-2 to pGE728-12 having a predetermined mutation in the AraR binding sequence (AraRBS) with respect to pGE728-1 were obtained. The Arar binding sequences in pGE728-1 to pGE728-12 are shown in No. 1 in Table 1 below. It is as shown in 1-12.

(11) LacZ assay (expression of β-galactosidase)
For pGE728-1 to pGE728-12 prepared as described above, the controllability of gene expression was evaluated according to the procedure of the β-galactosidase gene (LacZ) reporter assay described in the above embodiment. In the induction of expression in the procedure (1) of the LacZ reporter assay, the final concentration of arabinose in the medium was set to 2%, and arabinose was added to the culture medium in the range of OD ₆₁₀ = 0.5 to 1. In addition, in the procedure (2), after culturing for 5 hours, sampling was performed, and the activity of LacZ was measured for the obtained sample.
[result]
The results of Test Example 1 are shown in Table 1 below.

As shown in Table 1, sample No. Sample Nos. 9 to 12 in which Arar binding sequences (SEQ ID NOs: 5 to 12) having a predetermined nucleotide sequence were combined with Ptac, respectively. Regarding 1 to 8, it was shown that the promoter activity was induced 10 times or more higher when arabinose was added than when arabinose was not added. Above all, sample No. Sample Nos. 2, 3, 4, and 6 have relatively high promoter activity induction efficiencies, and in particular, Ptac combined with SEQ ID NOs: 2 and 3. Nos. 2 and 3 showed that the promoter activity increased more than 80 times when arabinose was added to that when arabinose was not added.
In addition, sample No. Regarding 1 to 6, when arabinose was not added, the absolute value indicating the promoter activity was 10 or less, and it was confirmed that the promoter activity (LacZ expression) was strictly suppressed. Further, among these, the sample No. which is a combination of Ptac and SEQ ID NOs: 2 and 3. Regarding 2 and 3, when arabinose was not added, the absolute value indicating the promoter activity was less than 1, and it was confirmed that the promoter activity (LacZ expression) was particularly remarkably suppressed. That is, the sample No. which is a combination of Ptac and SEQ ID NO: 2 or 3. Regarding 2 and 3, when arabinose was not added, the promoter activity (LacZ expression) could be suppressed remarkably, and at the same time, the promoter activity was remarkably increased by the addition of arabinose, and a large amount of the target gene product in the active form was produced. It has been shown that it can be expressed. Therefore, the combination of Ptac with the nucleotide sequence of SEQ ID NO: 2 or 3 enables particularly strict control of gene expression (switching on and off) and is particularly high when gene expression is switched on. It can be said that this is a particularly preferable embodiment in the present invention in that the expression level can be secured.
As described above, it was shown that the predetermined sequence structure that the nucleic acid according to the present invention can have enables efficient and precise gene expression control by the addition / addition of arabinose.

[Test Example 2]
Two plasmid vectors, pGE728-13 and pGE728-14, were further introduced into the pGE728-1 constructed in Test Example 1 by introducing a predetermined mutation into the ArarR binding sequence by the same point mutation introduction method as in Test Example 1. Obtained. The Arar binding sequences in pGE728-13 and pGE728-14 are as shown in Table 2 below, respectively.
The following changes (i) and (ii) have been made to some of the plasmid vectors prepared in Test Example 1 (see Tables 1 and 2) and the newly prepared pGE728-13 and pGE728-14 in this Test Example. The controllability of gene expression was evaluated by the β-galactosidase gene (LacZ) reporter assay in the same manner as in Test Example 1.

(i) When the OD (turbidity) value of the culture solution was in the range of 0.5 to 1.5, arabinose was added to the culture solution to induce gene expression.
(ii) The promoter activity was evaluated by the activity ratio of β-galactosidase of the sample with arabinose induction to the sample without induction with arabinose as in Test Example 1, and the number of samples (n) shown in Table 2 for each plasmid vector. The test was carried out according to (number), and for plasmid vectors having n or more n numbers, an average value was calculated for the activity ratio of β-galactosidase in each sample, and the value of the average value was used as the promoter activity ratio.

Table 2 shows the results of Test Example 2.

As shown in Table 2, pGE728-2 and pGEE728-3 (which adopted SEQ ID NOs: 2 and 3 as AraRBS, respectively), which were confirmed to exhibit excellent gene expression control ability in Test Example 1, were also used in this Test Example. The promoter activity ratios were 94.1 and 98.9, respectively, confirming that they possessed excellent gene expression control ability.
Of particular note in the results of this test example, among the newly constructed pGE728-13 and pGE728-14, pGE728-13 showed a promoter activity ratio of 162, and the above-mentioned pGE728-2 and pGEE728-3 It was confirmed that the value of the promoter activity ratio was far exceeded and that the gene expression control ability was extremely excellent. In addition, it was confirmed that the pGE728-14 newly constructed in this test example retains sufficient gene regulation expression ability, although the promoter activity ratio is a relatively low value of 14.8. ..

Regarding pGE728-1, pGE728-6, pGE728-7, and pGE728-8, relatively large fluctuations were observed with respect to the promoter activity ratio obtained in Test Example 1. Although the cause is not clear, it is a matter predicted in the field of biology or biotechnology that it fluctuates depending on various conditions such as the timing of gene expression induction. Based on the results of Test Example 1 and this Test Example, pGE728-1 , PGE728-6, pGE728-7, and pGE728-8 can be fully understood that they have been reproduced to have excellent gene control ability.

As described above, the gene expression control sequence capable of exerting exceptionally excellent gene expression control ability was obtained by this test example, and the gene expression control sequence prescribed in the present invention controls gene expression in a reproducible and precise manner. It was shown that it can be done.

[Test Example 3]
In Test Example 3, the expression of the target gene (reporter gene) was controlled by using a coryneform bacterium in which the araE gene and the araR gene were integrated into the genome instead of the plasmid vector, and using an expression vector containing the gene control sequence according to the present invention. An example is shown.
The details of the test procedure are shown below.
(1) Preparation of expression plasmid vector By cleaving pGE728 with restriction enzymes KpnI and XbaI, a DNA fragment from which the araE coding region and the araR coding region had been removed was obtained. This DNA fragment was blunted with T4 DNA polymerase (Takara Bio), terminally phosphorylated with T4 polynucleotide kinase (Takara Bio), and then ligated with T4 DNA ligase (NEB) for cyclization. The resulting plasmid was named pGE716.
The structure of pGE716 is shown in FIG. 1 (b) and its nucleotide sequence is shown in SEQ ID NO: 81.

(2) Preparation of corine-type bacteria in which the araE gene and the araR gene are incorporated into the chromosome The chromosome gene recombination of corinebacterium glutamicum was generally carried out by a markerless gene recombination method using the sacB gene derived from bacillus. J. Mol. Microbiol. Biotechnology. 2004, 8: 243-254).
The details of the procedure are shown below.

(2-1) Construction of pGE015 (KanR / sacB / pUCori) pHSG299 (Takara Bio) is cleaved with BamHI and PstI, and the sacB fragment amplified by the PCR method is cleaved with the same restriction enzyme and ligated in a circular manner. To construct the plasmid vector pGE015.
For amplification of the sacB fragment by the PCR method, pNIC28-Bsa4 (Source BioScience in the United Kingdom; obtained from Danaform, a Japanese distributor) was used as a template. The nucleotide sequence of the amplified sacB fragment is shown in SEQ ID NO: 82.

(2-2) Construction of pGE209 (KanR / sacBm / pUCori) Using the following primers 1 and 2, the KpnI site was eliminated by introducing a mutation into the KpnI site existing in the SacB region of pGE015 by the PCR method. .. As a result, a plasmid vector pGE209 having a SacB gene (sacBm) coding sequence in which the KpnI site had disappeared was obtained.
Primer 1: 5'-TGAACAGATACCATTTGCCGTTCATT-3'(SEQ ID NO: 83)
Primer 2: 5'-AATGGTATCTGTTCACTGACTCCCCGC-3'(SEQ ID NO: 84)

(2-3) Construction of pGE285 (KanR / sacBm / pSC101ori) The pSC101ori fragment at the origin of replication of Escherichia coli was amplified by the PCR method in the same manner as in the procedure shown in Example 1.
Furthermore, pGE209 was used as a template to amplify a DNA fragment containing the KanR and sacBm regions. The nucleotide sequence of the amplified DNA fragment is shown in SEQ ID NO: 85.
The plasmid vector pGE285 was obtained by cyclically linking the above pSC101ori fragment and a DNA fragment containing the KanR and sacBm regions using an In-Fusion HD Cloning Kit.

(2-4) Construction of pGE825 (KanR / tnp1 upstream region-tunp1 downstream region / sacBm / pSC101ori) Corynebacterium glutamicum ATCC13032 strain A DNA fragment containing an upstream region and a downstream region of tunp1a, which is a transposase in the genome, was filed. A human-owned plasmid vector was used as a template and amplified by the PCR method. The nucleotide sequence of the amplified DNA fragment is shown in SEQ ID NO: 86.
Furthermore, by cleaving pGE285 with EcoRI, a linear DNA fragment cleaved between KanR and sacBm was obtained.
The plasmid vector pGE825 was obtained by cyclically linking the amplified fragment containing the upstream and downstream regions of tunp1a with the linear DNA fragment of pGE285 using an In-Fusion HD Cloning Kit.

(2-5) Construction of pGE837 (KanR / tnp1 upstream area-Ptac-araE-rrnBter-tunp1 downstream area / sacBm / pSC101ori) By cutting pGE825 with XhoI, cutting between the tnp1 upstream area and the tnp1 downstream area. The linear DNA was obtained.
Furthermore, a DNA fragment (Ptac-araE-rrnBter amplification fragment) in which Ptac, the araE gene coding region existing in the genome of the Corynebacterium glutamicum ATCC31831 strain, and the rrnBter terminator are linked in this order is used as a plasmid vector owned by the applicant. It was amplified by the PCR method as a template. The nucleotide sequence of the Ptac-araE-rrnBter amplified fragment is shown in SEQ ID NO: 87.
Then, using the In-Fusion HD Cloning Kit, the linear DNA fragment of pGE825 and the Ptac-araE-rrnBter amplification fragment were cyclically linked to obtain a plasmid vector pGE837.
The structure of pGE837 is shown in FIG. 2 (a), and its nucleotide sequence is shown in SEQ ID NO: 88.

(2-6) Introduction of pGE837 into Ptac-araE into the Corynebacterium glutamicum genome was introduced into the Corynebacterium glutamicum ATCC13032 strain by electroporation, and Δtnp1a :: Ptac-araE was introduced by the above markerless gene recombination method. A strain was prepared. The obtained strain was named GES759.

(2-7) Construction of pGE234 (KanR / tmp3b upstream region-PldhA-lacZ-rrnBter-tunp3b downstream region / sacBm / pUCori) The pGE209 was cleaved between KanR and SacBm by cutting with KpnI and BamHI. A linear DNA fragment was obtained.
Furthermore, the upstream region (tmp3b upstream region fragment) and the downstream region (tunp3b downstream region fragment) of the transposase tunp3b in the Corynebacterium glutamicum ATCC13032 genome were amplified by the PCR method, respectively. The nucleotide sequences of these amplified regions are shown in SEQ ID NOs: 89 and 90, respectively.
Further, using the pGEK020 constructed in Test Example 1 as a template, a fragment containing the region of PldhA-lacZ-rrnBter (PldhA-lacZ-rrnBter fragment) was amplified by the PCR method. This fragment was inserted in the process of preparing various vectors by the inventor, but as described below, in pAra1 used for finally introducing the araR gene into the coryneform bacterial genome, PldhA and The lacZ region has been removed as a result. Therefore, the information on the nucleotide sequence of the amplified PldhA-lacZ-rrnBter fragment is omitted.

The linear DNA of pGE209, the upstream fragment of tnp3b, the downstream fragment of tmp3b, and the PldhA-lacZ-rrnBter fragment are ligated in a circular manner using an In-Fusion HD Cloning Kit, and the plasmid vector pGE234 is linked. Was acquired.

(2-8) Construction of pAra1 (KanR / tnp3b upstream region-Ptac-araR-rrnBter-tunp3b downstream region / sacBm / pUCori) A linear chain in which the PldhA-lacZ region was removed by cutting pGE209 with KpnI and BamHI. A DNA fragment was obtained.
Furthermore, the Ptac fragment (SEQ ID NO: 91) was amplified by the PCR method using pGEK094 as a template.
Furthermore, the araR gene fragment (SEQ ID NO: 92) was amplified by the PCR method using pGE689 as a template.
Next, the linear DNA of pGE209, the Ptac fragment, the araR gene fragment, and the In-Fusion HD Cloning Kit were used to ligate them in a circular manner to obtain a plasmid vector pAra1.
The structure of pAra1 is shown in FIG. 2 (b), and its nucleotide sequence is shown in SEQ ID NO: 93.

(2-9) Introduction of Ptac-araR into the GES759 genome pAra1 was introduced into GES759 by electroporation, and a Δtnp3b :: Ptac-araR strain was prepared by the above markerless gene recombination method. The obtained strain was named AIS1.

(3) Transformation of AIS1 by pGE716 and LacZ assay (expression of β-galactosidase)
Then, pGE716 was electroporated into AIS1 and selected with kanamycin.
The resulting transformant was inoculated so that the liquid medium (4% Glc, 25μg / mL K an) in 10mL to OD610 = 0.2, 33 ℃, were cultured in 200 rpm. After culturing for 4 hours, half the volume of the culture solution (4.5 mL) was taken in another test tube and mixed with 0.5 mL of 20% arabinose to give a final concentration of arabinose of 2%. In addition, as a comparative control, a product without arabinose added was also prepared. After further culturing these samples for 2 hours, the culture solutions with and without arabinose induction were sampled, and the promoter activity was measured by the LacZ assay.
The procedure of the LacZ assay and the composition of the liquid medium are as described in the embodiment.

[result]
The results of the LacZ assay are shown in FIG.

First, the outline of Test Example 3 will be described. Test Example 3 is different from Test Example 1 in the following points.
That is, in Test Example 1, a plasmid vector encoding the araE gene and the araR gene in addition to the gene expression control sequence (R) and the reporter gene (LacZ gene) prescribed in the present invention was used as the plasmid vector to be introduced into the ATCC13032 strain. .. On the other hand, in Test Example 3, the AIS1 strain (derived from ATCC13032 strain) introduced in advance so that the araE gene and the araR gene are forcibly expressed in the genomic DNA is used, and the araE gene is used as the plasmid vector to be introduced into the strain. And pGE716 from which the araR gene was removed was used.

In FIG. 3, AIS1 / pGE716-3 and AIS1 / pGE716-3 are samples derived from different transformant colonies, respectively.
Focusing on the fold (induction efficiency) of the promoter activity, it showed 67 times for AIS / p716-3 and 160 times for AIS / p716-4, and according to the gene expression control sequence prescribed in the present invention, it was effective by arabinose. It was shown that the expression of the target gene can be controlled.

[Test Example 4] (Effect of arabinose concentration / induction time)
Similar to Test Example 3, pGE716 was electroporated into strain AIS1 and selected with kanamycin to obtain a transformant.
The transformed cells were inoculated into 10 mL of a liquid medium so that OD610 = 1.0, and cultured at 33 ° C. and 200 rpm. After 4 hours, half the volume (5 mL) was taken in a separate test tube and 20% arabinose was added so that the final concentrations of arabinose in the medium were 0.002%, 0.02%, 0.2% and 2%, respectively. A sample to which arabinose was not added was also prepared as a comparative control.
The above samples were cultured, and the culture solutions with and without arabinose induction were sampled after 4 hours, 20 hours, and 24 hours, respectively, and the promoter activity was measured by the LacZ assay.
The procedure of the LacZ assay and the composition of the liquid medium are as described in Test Example 3.

[result]
The results of the LacZ assay are shown in FIG.
As shown in FIG. 4, the expression level had already reached a nearly saturated state 4 hours after the induction of expression by arabinose, and no significant increase in the expression level of the reporter gene was observed thereafter. That is, at least according to the sequence composition of the nucleic acid according to this test example, it was clarified that a sufficient expression level of the target gene can be obtained within 4 hours after the induction of expression.
Furthermore, regarding the arabinose concentration, 0.02% showed the highest expression level (promoter activity), but it was revealed that even the lowest concentration of 0.002% can induce a sufficient level of promoter activity.

[Test Example 5] (Combination with various promoters)
(1) Construction of pGEK035 (MCS-SD-lacZ-rrnBter / KanR / pUCori / pCG1ori) pGEK030 constructed in the section of Test Example 1 (4) was cleaved with restriction enzymes XhoI and NdeI to obtain pGEK030 cleavage fragments. .. An oligo DNA having the following sequence was annealed into this cleavage fragment and ligated to prepare a vector pGEK035 into which the SD sequence was inserted. This SD sequence has been removed in pAra24 to pAra30, which are the final constructs for which promoter activity has been confirmed.
SD sequence: 5'-tAATATTGAAAGGGAGGtt-3'(SEQ ID NO: 95)

(2) Construction of pGEK107 (MCS-SD-lacZ-rrnBter / KanR / pSC101ori / pCG1ori) By treating pGEK035 with restriction enzymes NotI and XmaI, the replication origin pUCori contained in pGEK035 was removed, and the origin of replication was defined as the origin of replication. It was replaced with the E. coli origin of replication pSC101ori (low copy count) contained in the applicant's own plasmid vector. The resulting vector was named pGEK107.

(3) Preparation of shuttle vector by combining various promoters and AraR binding sequence Using genomic DNA of Corinebacterium glutamicum ATCC13031 strain as a template, the constitutive promoters tuf, dapA, and sod promoters and inducible promoters were used by the PCR method. Certain ldhA, gapA, and metE promoter fragments were amplified, respectively. In addition, the plasmid vector pGE716 constructed in Test Example 3 was used as a template, and the ArarR binding sequence and the SD sequence were amplified by the PCR method.
Next, each promoter fragment obtained by the PCR method as described above and an amplified fragment of the ArarR-binding sequence and the SD sequence were ligated by the overlap PCR method. By this overlapping PCR method, DNA fragments having the nucleotide sequences shown in SEQ ID NOs: 96 to 102 were obtained. These DNA fragments contain the nucleotide sequences of SEQ ID NOs: 96 to 102 shown as the preferred embodiments of the nucleic acids of the present invention in the section of the above embodiments.

The pGEK107 obtained in the above section (2) was cleaved with restriction enzymes KpnI and NdeI, and each of the above promoter / Arar binding sequence fragments was circularized with In-Fusion HD Cloning Kit in the obtained vector fragment. Connected. The resulting plasmids were named pAra24, pAra25, pAra26, pAra27, pAra28, pAra29, and pAra30, respectively.

In the same manner as in Test Example 3, each plasmid was electroporated into the strain AIS1 and selected with kanamycin to obtain transformants, and the obtained transformants were placed in 10 mL of a liquid medium at OD610 = 0.2. The cells were inoculated in this manner and cultured at 33 ° C. and 200 rpm. After 4 hours, half the volume (5 mL) of the culture medium was taken in another test tube and 20% arabinose was added so that the final concentration of arabinose in the medium was 0.2%. In addition, as a comparative control, a product without arabinose added was also prepared. After further culturing these samples for 4 hours, the culture medium with and without arabinose induction was sampled, and the promoter activity was measured by the LacZ assay.
The procedure of the LacZ assay and the composition of the liquid medium are as described in Test Example 3.

[result]
The results of the LacZ assay are shown in Table 3 below.

As shown in Table 3, it was confirmed from this test example that gene expression can be strictly controlled by adopting not only the tac promoter but also various other promoters as the promoter site. In addition, for pAra30 in which the estimated transcription initiation site overlaps the AraR binding sequence in a predetermined embodiment, the gene expression control ability is superior to that of pAra24 in which the estimated transcription initiation site does not overlap with the AraR binding sequence. Indicated.

As described above, according to the present test example, according to the predetermined configuration of the present invention in which the AraR binding sequence having a predetermined nucleotide sequence is adopted as one element of the gene control sequence, the promoter site is not limited to the tac promoter. It has been demonstrated that excellent gene regulatory ability can be exerted even when various other promoters including a target promoter and an inducible promoter are adopted.

[Comparative Test Example 1]
Comparative Test Example 1 is a comparative example in which control of the target gene was attempted using the promoter region of the araE gene naturally occurring in the genome of the Corynebacterium glutamicum ATCC31831 strain instead of the control sequence according to the present invention.
The details of the test procedure are shown below.

(1) Construction of pGE728ParaE (PdapA-araE-rrnBT1ter-T7ter / PdapA-araR / ParaE-lacZ-rrnBter / KanR / pSC101ori / pCG1ori) The pGE728 is cleaved in the GPnI and NdeI regions by KpnI and NdeI. A linear DNA consisting of the removed sequence was obtained.
Furthermore, using the Corynebacterium glutamicum ATCC31831 strain genomic DNA as a template, a DNA fragment (ParaE fragment) containing the promoter region of the araE gene shown in FIG. 5 was amplified according to the PCR method. The nucleotide sequence of the amplified ParaE fragment is shown in SEQ ID NO: 94.
The plasmid vector pGE728ParaE was prepared by ligating the linear DNA and the ParaE fragment in a circular manner using a Gibson Assembly Cloning Kit.

In the procedure for obtaining the transformant shown in Test Example 1 and the LacZ assay, the transformant of Corynebacterium glutamicum ATCC13032 strain was similarly prepared except that the expression vector pGE728ParaE obtained as described above was used. Obtained and evaluated for promoter activity by LacZ assay.

[result]
The results of the LacZ assay are shown in Table 4 below.

As shown in Table 4, the activity value with arabinose induction (that is, with arabinose added) becomes lower than the activity value without arabinose induction (that is, without arabinose addition), and gene expression control by arabinose is impossible. Was shown to be.
The nucleotide sequence used as the gene control sequence in Comparative Test Example 1 is uniquely present on the genomic DNA of the Corynebacterium glutamicum ATCC31831 strain as shown in FIG. 5, and contains a natural AraR binding sequence. It's a waste. That is, even if the promoter region peculiar to the coryneform bacterium as shown in Non-Patent Document 6 is used, as shown in the results of Table 2, the gene expression control achieved by the nucleotide sequence prescribed in the present invention is not achieved. It was possible and did not even show promoter activity as a gene expression system in the first place.

(Supplementary information)
In the above test examples and comparative test examples, information on nucleotide sequences such as various gene coding regions, promoter regions, terminator regions, and origins of replication amplified by the PCR method in the process of constructing various plasmid vectors is disclosed in the sequence listing. Therefore, the sequence information of the primers used for PCR amplification was omitted. Those skilled in the art can refer to the information on each nucleotide sequence disclosed in the sequence listing or the information on the nucleotide sequence existing in the gene bank or the like to construct a plasmid vector or the like used in the above test example or the present invention. Nucleic acid and the like can be appropriately prepared.
In the above test examples and comparative test examples, there is also a step using In-Fusion HD Cloning Kit or Gibson Assembly Cloning Kit for cloning various gene coding regions, promoter regions, terminator regions, replication origins, etc. as described above. However, regarding the primer pair used for PCR amplification, it is supplemented that an appropriate adapter sequence is added to each of the 5'ends of the forward / reverse primer according to the instructions of the cloning kit.

The present invention has high industrial applicability in the fields of biotechnology, the field of producing substances such as chemical substances and biomaterials, and the like.

Claims (31)

A gene expression control sequence (R) containing at least a part of the nucleotide sequence (X) capable of functioning as a promoter and the nucleotide sequence (Y) described in (a) or (b) below.
Nucleic acid: The gene expression control sequence (R) satisfies the following condition (I).
(A) The nucleotide sequence according to any one of SEQ ID NOs: 5 to 12 and SEQ ID NOs: 17 and 18;
(B) A nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence according to (a) above.
However, when the nucleic acid is RNA, thymine (t) in the nucleotide sequence shall be read as uracil (u).
Condition (I): In a coryneform bacterium, the promoter activity of the nucleotide sequence (X) is suppressed when arabinose is not added. The nucleic acid according to claim 1, wherein the nucleotide sequence (Y) is directly or indirectly linked to the 3'end of the nucleotide sequence (X). The nucleic acid according to claim 1 or 2, wherein the nucleotide sequence (X) is a constitutive promoter. The nucleotide sequence (X) is trc promoter, tacI promoter, tacII promoter, T5 promoter, T7 promoter, lac promoter, trp promoter, tet promoter, EFTu promoter, groES promoter, SOD promoter, P15 promoter, ldhA promoter, gapA promoter, dapA. The nucleic acid according to any one of claims 1 to 3, which is selected from a promoter, a metE promoter, and a tuf promoter. The nucleotide sequence (X) is a promoter sequence derived from 5'upstream of a gene present in the genomic DNA of a coryneform bacterium, a promoter sequence derived from a plasmid inherent in the coryneform bacterium, or a phage capable of infecting the coryneform bacterium. The nucleic acid according to any one of claims 1 to 4, which comprises a promoter sequence derived from the genome. The gene expression control sequence (R) shows at least 10 times higher promoter activity in coryneform bacteria when arabinose is added than in the case where arabinose is not added, according to any one of claims 1 to 5. The nucleic acid according to item 1. The gene expression control sequence (R) shows at least 15 times higher promoter activity in coryneform bacteria when arabinose is added than in the case where arabinose is not added, according to any one of claims 1 to 6. The nucleic acid according to item 1. The gene expression control sequence (R) shows at least 20 times higher promoter activity in coryneform bacteria when arabinose is added than in the case where arabinose is not added, according to any one of claims 1 to 7. The nucleic acid according to item 1. The gene expression control sequence (R) shows at least 35 times higher promoter activity in coryneform bacteria when arabinose is added than in the case where arabinose is not added, according to any one of claims 1 to 8. The nucleic acid according to item 1. The gene expression control sequence (R) shows at least 50 times higher promoter activity in coryneform bacteria when arabinose is added than in the case where arabinose is not added, according to any one of claims 1 to 9. The nucleic acid according to item 1. The gene expression control sequence (R) shows at least 70 times higher promoter activity in coryneform bacteria when arabinose is added than in the case where arabinose is not added, according to any one of claims 1 to 10. The nucleic acid according to item 1. The nucleic acid according to any one of claims 1 to 11, wherein the nucleotide sequence (Y) is represented by the following general formula (I):
5'-N ₁ TGTN ₂ AGCGN ₃ TN ₄ AN ₅ N ₆ N ₇ -3'... General formula (I)
In the formula, N ₁ , N ₂ , N ₃ , N ₄ , N ₅ , N ₆ and N ₇ independently indicate A (adenine), G (guanine), C (cytosine) or T (thymine), respectively. Or, if it is deleted and the nucleic acid is RNA, T (thymine) shall be read as U (uracil). The nucleic acid according to any one of claims 1 to 12, wherein the gene expression control sequence (R) contains any of the following nucleotide sequences (l) to (o):
(L) The nucleotide sequence according to any one of SEQ ID NOs: 22 to 61 and SEQ ID NOs: 96 to 102;
(M) A nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence according to (l);
(N) A nucleotide sequence that hybridizes with a nucleotide sequence complementary to the nucleotide sequence described in (l) under stringent conditions; and a sequence of at least 80% or more of the nucleotide sequence described in (o) (l). Nucleotide sequences with identity,
However, when the nucleic acid is RNA, thymine (t) in the base sequence should be read as uracil (u). The nucleic acid according to any one of claims 1 to 13, wherein the gene expression control sequence (R) further contains an SD sequence. The nucleic acid according to any one of claims 1 to 14, wherein the SD sequence is directly or indirectly linked to the 3'end of the nucleotide sequence (Y). The nucleic acid according to any one of claims 1 to 15, further comprising a nucleotide sequence encoding at least one of an araE protein and an araR protein. The nucleic acid according to any one of claims 1 to 16, which comprises a nucleotide sequence encoding at least one of an araE protein and an araR protein derived from a coryneform bacterium. The nucleic acid according to any one of claims 1 to 17, further comprising one or more nucleotide sequences selected from the group consisting of an origin of replication, a selectable marker gene, a cloning site, a restriction enzyme recognition site, and a terminator. The nucleic acid according to any one of claims 1 to 18, which is provided as a plasmid capable of autonomous replication in bacteria. The nucleic acid according to any one of claims 1 to 19, which is provided as an expression vector. The nucleotide sequence (Z) further contains a nucleotide sequence (Z) encoding the target gene, and the nucleotide sequence (Z) is a gene expression control sequence (R) such that the expression of the target gene is controlled by the gene expression control sequence (R) in coryneform bacteria. ) Is directly or indirectly linked to the nucleic acid according to any one of claims 1 to 18. The nucleic acid according to any one of claims 1 to 21, which is DNA. A bacterium into which the nucleic acid according to any one of claims 1 to 22 has been introduced. The bacterium according to claim 23, which is a bacterium belonging to the genus Escherichia. The bacterium according to claim 23, which is a coryneform bacterium. A DNA having at least one nucleotide sequence encoding an araE protein and an araR protein derived from a coryneform bacterium is incorporated into the genomic DNA, and the genomic DNA is configured to be capable of expressing the araE protein and the araR protein. The bacterium according to any one of claims 23 to 25. A method for expressing a target gene, which comprises expressing the target gene by exposing the bacterium according to any one of claims 23 to 26 to arabinose or an analog thereof. 27. The method of claim 27, wherein the bacterium is a coryneform bacterium. A method for producing a target substance, which comprises expressing the target gene by exposing the bacterium according to any one of claims 23 to 26 to arabinose or an analog thereof. 29. The method of claim 29, wherein the bacterium is a coryneform bacterium. A nucleic acid fragment for use in controlling the expression of a target gene in a coryneform bacterium.
Nucleic acid fragment containing the nucleotide sequence (Y) according to (a) or (b) below:
(A) The nucleotide sequence according to any one of SEQ ID NOs: 5 to 9, 11 and 12 and SEQ ID NOs: 17 and 18;
(B) A nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence according to (a) above.
However,
When the nucleic acid is RNA, thymine (t) in the nucleotide sequence shall be read as uracil (u).
It is assumed that the nucleotide sequences shown in SEQ ID NOs: 10, 20 and 21 as the nucleotide sequence (Y) are excluded, and the nucleotide sequence (Y) satisfies the following condition (II).
Condition (II): Corine-type bacterium transformed with a plasmid vector in which the nucleotide sequences from the 2913th to the 2928th positions in the nucleotide sequence of the plasmid vector pGE728-1 shown in SEQ ID NO: 80 are replaced with the above nucleotide sequence (Y). In, the promoter activity of PtacI is suppressed when arabinose is not added as opposed to when arabinose is added.