JP6991897B2 - Nucleic acids and vectors for controlling gene expression in non-phototrophic C1 metabolizing microorganisms, and transformants thereof - Google Patents

Description

The present invention relates to a method for controlling gene expression in a microorganism. More specifically, the present invention relates to nucleic acid molecules necessary for controlling gene expression in non-photonourishable C1 metabolizing microorganisms, particularly to the production of substances from methane and methanol using microorganisms.

Technology for producing useful substances such as chemicals and proteins from plant resources such as glucose and cellulose by metabolically modified microorganisms is being developed and put into practical use. Most of the conventional techniques have been made from organic substances, especially sugar, but by using modified phototrophic C1 metabolizing microorganisms, substance production technology from alternative raw materials such as methane and methanol has attracted new attention in recent years. It became so.

In order to impart the production capacity of substances that cannot be produced by nature to the phototrophic C1 metabolizing microorganisms used as the host, it is necessary to introduce genes encoding foreign enzymes required for production into the host cells and overexpress them. be. Despite this potential value, the types and numbers of substances that can be produced are still limited. One of the main causes is that it is often difficult to introduce an enzyme gene for substance production into a C1 metabolizing microorganism. In general, the growth of C1 metabolizing microorganisms is slower than that of heterotrophic bacteria. Therefore, when the product of the expressed gene competes with growth, the basal expression level at the time of non-induction is infinite not only to maintain the genotype stably for a long period of time but also to obtain a transformant in the first place. It is desirable to keep it low. Therefore, a technique that can induce gene expression only under desired conditions is very important. On the other hand, in order to maximize substance productivity, it is also strongly required to be able to strongly induce gene expression at any timing. That is, there is a strong demand for a technique that can strongly induce expression at the time of induction while the basal expression is extremely small.

In addition, there are still many unclear points about the biological properties of phototrophic C1 metabolizing microorganisms, and it is desired to accumulate basic knowledge. Therefore, even in the elucidation of the physiological function of a specific gene, it is expected that the induction of gene expression limited to desired conditions will greatly contribute to the promotion of research using phototrophic C1 metabolizing microorganisms.

Despite the strong demands mentioned above, there is a marked lack of gene expression control tools for non-phototrophic C1 metabolizing microorganisms compared to heterotrophic bacteria that have been widely used as gene recombination hosts (non-phototrophic C1 metabolizing microorganisms). Patent Document 1). One of the major causes is that many of the gene expression control systems commonly used in heterotrophic bacteria such as Escherichia coli cannot be diverted to C1 metabolizing microorganisms (Patent Documents 2 and 3). For example, Patent Document 1 describes that an isopropylthiogalactopyranoside (IPTG) -inducible trc promoter is used, but it does not show good performance in methane-utilizing bacteria. In fact, except for the examples of Patent Document 1 and Non-Patent Document 2, the expression control systems reported so far have been identified from the genomes of non-phototrophic C1 metabolizing microorganisms (Patent Documents 2 and 3). In addition, none of the above prior arts can achieve both low basal expression and strong expression induction at the same time.

As described above, although an expression control system that functions well in non-phototrophic C1 metabolizing microorganisms has been strongly desired, there have been no such reports. The reason was that the existing expression control system could not be diverted to non-phototrophic C1 metabolizing microorganisms, and instead, those identified from non-phototrophic C1 metabolizing microorganisms had limited performance.

For example, Patent Document 2 and Non-Patent Document 2 describe inducible promoters that can be used in methane-utilizing bacteria, but not only have basal expression, but also the expression ratio between non-induction and induction. Was also limited. In Patent Document 2, the inducible promoter sequence is referred to as Methylomonas sp. Although found from the genome of, there is a problem that expression induction is limited to specific nutritional conditions. Finally, in Bacillus metabolism MGA3 strain (Non-Patent Document 3) and Pichia pastoris, which are methanol-utilizing bacteria, examples of use of a mannitol-induced promoter derived from the same species, a xylose-induced promoter derived from a related species, and a methanol-induced promoter are used. However, its use in other non-photonutrient C1 metabolizing microorganisms has not been shown. In fact, mannitol, xylose, and methanol-inducible promoters depend on complex expression control via a signal transduction system unique to the host microorganism, which makes it difficult to apply to non-phototrophic C1 metabolizing microorganisms that do not have this. Conceivable.

As described above, as far as the present inventors know, the expression control systems for non-phototrophic C1 metabolic microorganisms known so far are not only limited in their performance but also naturally derived or modified thereof. Most of them showed their use in specific microbial species.

Patent Document 1: US 2014/273128 Publication Patent Document 2: WO2015 / 195972 Pamphlet Patent Document 3: Japanese Patent Application Laid-Open No. 2006-515166

Non-Patent Document 1: Kalyuzhnaya, M. et al. G. et al. , Metabolic engineering in methanotropic bacteria. , Metab. Eng. , Vol. 29,142-152 (2015)
Non-Patent Document 2: Hender, C.I. A. et al. , Biological conversion of methane to lacticate by an obligate methanotrophic bacteria. , Sci. Rep. , Vol. 6,21585 (2016)
Non-Patent Document 3: Irla, M. et al. et al. , Genome-based genetic tool develop for Bacillus methanolicus: theta-and rolling circle-replicating plasmids for index , Front. Microbiol. , Vol. 7,1481 (2016)
Non-Patent Document 4: Salis, H. et al. M. , The ribosome bidding site calculator. , Methods Enzymol. , Vol. 498, 19-42 (2011)

In view of the above situation, an object of the present invention is to provide a technique that can be widely used in non-phototrophic C1 metabolizing microorganisms, has a negligibly small basal expression, and has a large expression level ratio between non-induction and induction. It is in. In particular, an object of the present invention is to provide a technique capable of achieving protein expression in an amount sufficient for improving the amount of substance produced from a C1 raw material at an arbitrary timing so as not to compete with cell growth.

As described above, it is generally recognized that most of the gene expression control systems used in heterotrophic bacteria such as Escherichia coli cannot be diverted to non-phototrophic C1 metabolizing microorganisms (Patent Document 3). It was not found that the expression induction system by riboswitch can also be used for non-phototrophic C1 metabolizing microorganisms (Patent Document 1). However, the present inventors have conducted diligent studies to solve the above-mentioned problems, and have found that the expression induction system by riboswitch functions particularly well in many non-phototrophic C1 metabolizing microorganisms. Specifically, this system has features that are not found in the expression control system in conventional non-phototrophic C1 metabolizing microorganisms, in which the basal expression is extremely small and the expression ratio at the time of non-induction and at the time of induction is significantly larger than that of the prior art. It was found that the present invention was completed. In particular, when the expression induction system of the present invention is applied to methane-utilizing bacteria, not only basal expression is not detected at all, but also strong expression induction is possible, and it has been found that it has features that cannot be achieved by Escherichia coli. rice field.

In addition, the present inventors have previously attempted to introduce a vector containing a sequence that constitutively expresses the L-lysine decarboxylase gene into a non-phototrophic C1 metabolizing microorganism, but obtain a completely transformant. Was not done. Therefore, based on the above-mentioned new finding that the basal expression is extremely small in the expression system using the riboswitch, the present inventors replaced the constitutive expression system with the expression induction system using the riboswitch. It has been found that the acquisition efficiency of transformants can be dramatically improved. Since a transformant that constitutively expresses GFP protein that does not affect the growth of cells can be easily obtained, an expression-inducing system is particularly used when expressing a gene that competes with the growth of non-photonourishable C1 metabolizing microorganisms. It proved that the utilization of was very important.

That is, the present invention provides:
(1) The nucleic acid for controlling gene expression in a non-phototrophic C1 metabolizing microorganism, which comprises a sequence encoding a riboswitch;
(2) The nucleic acid of (1) above, wherein the riboswitch is a theophylline-binding riboswitch;
(3) The nucleic acid of (2) above, characterized in that the theophylline-binding riboswitch has the sequence of 5'-GGTACC GGTGATACCAGCATCCGTGTGATCTCTTGGCAGCACC CTGCTAAGGAGGCAACAAG-3'(SEQ ID NO: 1);
(4) The nucleic acid according to any one of (1) to (3) above, wherein the riboswitch is located downstream of a natural or artificial promoter;
(5) The nucleic acid of (4) above, wherein the promoter is an inducible promoter;
(6) The nucleic acid of (4) above, wherein the promoter is a homeostatic promoter;
(7) The nucleic acid of the above (5) or (6), wherein the promoter is a trc promoter;
(8) Nucleic acid according to (1) to (7) above, which comprises a natural or artificial ribosome binding sequence;
(9) The nucleic acid of (8) above, wherein the ribosome-binding sequence has a sequence of 5'-AAGGAGG-3';
(10) The nucleic acid of (8) or (9) above, wherein the ribosome binding sequence is located downstream of the riboswitch via a spacer;
(11) The nucleic acid according to any one of (1) to (10) above, which further comprises a gene to be expressed, wherein the gene is located upstream of the terminator;

(12) A vector containing any of the nucleic acids (1) to (11) above;
(13) A transformant of a non-phototrophic C1 metabolizing microorganism containing any of the nucleic acids (1) to (11) above;
(14) The transformant of (13) above, wherein the nucleic acid is integrated on the chromosome of the host microorganism;
(15) The transformant of (13) above, wherein the nucleic acid has been introduced into a host microorganism as a vector containing the nucleic acid;
(16) The transformant according to any one of (13) to (15) above, wherein the non-phototrophic C1 metabolizing microorganism is a methane-utilizing bacterium or a methanol-utilizing bacterium;
(17) Non-phototrophic C1 metabolizing microorganisms include Methylomonas, Methylococcus, Methylococcus, Methylocystis, Methylocystis, Methylocystis, Methylocystis. , One of (13) to (15) above, characterized in that it is selected from the group consisting of Methylocystis, Methylobacterium, Xanthobacter and Paracoccus. Transformant;
(18) Non-phototrophic C1 metabolizing microorganisms are Methylococcus transformus Bath (NCIMB 11132 ), Methylophilus methylophilus AS1 (NCIMB 10515), Methylococcus Cepsculus Characteristically, the transformant according to any one of (13) to (15) above;

(19) A method for inducing expression of a gene under the control of a riboswitch by supplying a ligand for the riboswitch to any of the transformants (13) to (18) above;
(20) The method of (19) above, wherein the ligand is theophylline;
(21) The method of (19) or (20) above, wherein the ligand is autonomously supplied by the transformant;
(22) A kit for transformation of a non-phototrophic C1 metabolizing microorganism containing the nucleic acid according to any one of (1) to (11) above.

Since the basal expression can be suppressed to an extremely small value by the present invention, it is easy to obtain a nucleic acid transformant containing a gene that competes with growth, which was not obtained in the conventional constitutive expression or an expression-inducing system in which the basal expression cannot be ignored. can do. That is, by using the expression-inducing system of the present invention, the types of substances that can be produced by non-phototrophic C1 metabolizing microorganisms can be significantly expanded. Furthermore, the present invention has a feature that the expression ratio at the time of non-induction and at the time of induction is significantly larger than that of the prior art using an inducer such as IPTG. It is possible to maximize the performance of substance production and elucidate the physiological properties of genes through such regulation.

FIG. 1 is a plasmid map of pmk61A. In the figure, "Ptrc" is the trc promoter, "riboswitch" is the riboswitch sequence, "RBS" is the ribosome binding sequence, "GFP" is the sfGFP gene, "mob" is the mob gene, and "kanR" is kanamycin. The resistance gene and "Rep] indicate the origin of replication, respectively. FIG. 2 is a graph showing the relationship between theophylline addition and GFP fluorescence intensity in Escherichia coli introduced with pmk61A. FIG. 3 is a graph showing the relationship between theophylline addition concentration and GFP fluorescence intensity in Methylococcus capsulatus Bath introduced with pmk61A. FIG. 4 is a graph showing the relationship between theophylline addition and GFP fluorescence intensity in Methylophilus methylophilus AS1 introduced with pmk61A . FIG. 5 is a graph showing the relationship between theophylline addition and GFP fluorescence intensity in Methylobacillus glycogenes into which pmk61A has been introduced. FIG. 6 is a graph showing the relationship between theophylline addition and GFP fluorescence intensity in Paracoccus denitrificans introduced with pmk61A. FIG. 7 is a plasmid map of pmk96 and pmk99. In the figure, "Ptrc" is the trc promoter, "riboswitch" is the riboswitch sequence, "RBS" is the ribosome binding sequence, "cadA" is the L-lysine decarboxylase (cadA) gene, and "mob" is the mob. The gene is indicated by "kanR", the canamycin resistance gene is indicated by "Rep", and the origin of replication is indicated by "Rep".

The present invention will be specifically described below.

In the present invention, nucleic acid refers to ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), wherein the nucleic acid of the present invention contains a sequence encoding a riboswitch.

A riboswitch is an RNA that selectively binds to a specific small molecule compound, and the small molecule compound is called a ligand. In the absence of a ligand, it forms a secondary structure with RNA base pairs and affects the nucleic acids around the riboswitch. In particular, when a ribosome binding site is contained downstream of the riboswitch, it prevents the ribosome from approaching the ribosome binding site, thereby hindering the translation of the mRNA of the gene located downstream thereof. On the other hand, in the presence of a ligand, the ribosome can approach the ribosome binding site through the elimination of the secondary structure associated with the ligand binding. Therefore, the mRNA of the gene is translated only when the ligand is added, and the expression of the target gene is induced.

The ligand in the present invention is not particularly limited as long as it can eliminate the secondary structure of the riboswitch in the absence of the ligand, and is naturally derived, modified thereof, or artificially modified. You can select one or any of the above.

The riboswitch in the present invention is not particularly limited, and may be any RNA that causes a secondary structure change due to ligand binding. For example, theophylline responsive riboswitch, adenosylcobalamine responsive riboswitch, cyclic di GMP responsive riboswitch, flavin mononucleotide responsive riboswitch, glucosamine-6-phosphate responsive riboswitch, glutamine responsive riboswitch, Glycin-responsive riboswitch, lysine-responsive riboswitch, preQ1 _- responsive riboswitch, purine-responsive riboswitch, S-adenosylhomocysteine-responsive riboswitch, S-adenosylmethionine-responsive riboswitch, S-adenosyl Examples include, but are not limited to, homocysteine & S-adenosylmethionine responsive riboswitches, tetrahydrofolic acid responsive riboswitches, thiaminepyrophosphate responsive riboswitches, molybdenum responsive riboswitches, and adenin responsive riboswitches. It may be known to already exist in nature, newly isolated from nature, artificially designed, or may be one or a combination of two or more.

The riboswitch in the present invention is preferably a theophylline-responsive riboswitch. The theophylline-responsive riboswitch is not particularly limited as long as the RNA secondary structure in the absence of theophylline is eliminated by theophylline binding. For example, 5'-GGTACC GGTGATACCAGCATCGTCTTGATGCCCTTGGCAGCACC CTGCTAAGGAGGCAACAAG-3 '( SEQ ID NO: 1), 5'-GGTACC GGTGATACCAGCATCGTCTTGATGCCCTTGGCAGCACC CTGAGAAGGGGCAACAAG-3' ( SEQ ID NO: 2), 5'-GGTACC GGTGATACCAGCATCGTCTTGATGCCCTTGGCAGCACC CGCTGCGCAGGGGGTATCAACAAG-3 '( SEQ ID NO: 3), 5'-GGTACCTGATAAGATAGG GGTGATACCAGCATCGTCTTGATGCCCTTGGCAGCACC AAGGGACAACAAG-3 ' ( SEQ ID NO: 4), 5'-GGTACC GGTGATACCAGCATCGTCTTGATGCCCTTGGCAGCACC CTGCTAAGGTAACAACAAG-3' ( SEQ ID NO: 5), 5'-GGTACC GGTGATACCAGCATCGTCTTGATGCCCTTGGCAGCACC CTGCTAAGGAGGTAACAACAAG-3 '( SEQ ID NO: 6) sequence, such as However, the present invention is not limited to these. More preferably, it has the sequence of 5'-GGTACCGGCATCGTCTTGATTGCCCTTGGCAGCACC CTGCTAAGGAGGCAACAAG -3'(SEQ ID NO: 1).

Moreover, theophylline which is a ligand may be a derivative of theophylline. The concentration of theophylline or a derivative thereof added to the culture of the transformant of the present invention as a ligand is preferably 0.1 to 10 mM.

The riboswitch in the present invention is located downstream of a natural or artificial promoter, thereby operably linked to that promoter. The promoter is DNA located upstream of the expressed gene and contains a promoter sequence that attracts the RNA polymerase of the host microorganism to the template DNA.

The promoter in the present invention is not particularly limited as long as it operates in a non-phototrophic C1 metabolizing microorganism. Therefore, the nucleic acid is not limited to a nucleic acid derived from a non-phototrophic C1 metabolizing microorganism, and may be a naturally occurring nucleic acid, or an artificially designed or modified nucleic acid. For example, methanol dehydrogenase promoter, methanemonooxygenase promoter, ribosome protein promoter, hexulose-6-phosphate synthase promoter, phosphohexulose isomerase promoter, glyceraldehyde-6-phosphate dehydrogenase promoter, rhaBAD promoter, araBAD promoter, tet promoter. , Trc promoter, tac promoter, trp promoter, lac promoter, lacUV5 promoter, lux promoter, phoA promoter, T7 promoter, λ phage PR promoter, PL promoter and the like. Further, as the promoter of the present invention, either homeostasis or inducibility can be selected depending on the expression level and properties to be achieved, and one or a plurality of promoters can be combined. An example of a preferred promoter in the present invention is the trc promoter.

The nucleic acid in the present invention comprises a ribosome binding sequence. Ribosome-binding sequences are nucleic acids, generally 4 to 10 bases in length, required to bind ribosomes to mRNA and initiate translation. It has characteristics rich in adenine or guanine, but is not particularly limited as long as it can operate in a non-phototrophic C1 metabolizing microorganism. Therefore, the ribosome-binding sequence of the present invention may be a naturally occurring sequence or an artificially designed sequence, and a suitable sequence can be selected according to the expression level and properties desired to be achieved. For example, as shown in Non-Patent Document 3, it can be arbitrarily designed according to the gene expression level to be achieved. Preferred examples of the ribosome-binding sequence of the present invention include a sequence having 5'-AAGGAGG-3'. These ribosome-binding sequences are typically located downstream of the riboswitch of the invention, thereby operably linked to the riboswitch. Therefore, a preferred embodiment of the invention is a nucleic acid in which the riboswitch is located downstream of a natural or artificial promoter and the ribosome binding sequence is located downstream of the riboswitch.

In addition, in this invention, "operably linked" includes both the case where both sequences are directly linked and the case where both sequences are linked via an appropriate number of bases. For example, a spacer consisting of a surplus sequence found in the vicinity of a known promoter and an appropriate number of bases may be present between the promoter and the riboswitch. Further, the ribosome binding sequence and the riboswitch may be linked via a spacer consisting of an appropriate number of bases. The length of the spacer can typically be 1-20 bases, preferably 5-10 bases.

It is also a preferred embodiment that the nucleic acid in the present invention contains a gene to be expressed. The gene in the present invention may be any gene that can be expressed by a non-phototrophic C1 metabolizing microorganism such as a marker gene, a fluorescent protein gene, a pigment biosynthesis gene, a gene belonging to a substance production pathway, and a gene belonging to a carbon metabolism system. You can select one or more. When expressing a plurality of different genes, for example, a plurality of units for each gene consisting of a riboswitch, a ribosome-binding sequence, and a gene may be arranged, and each of these units may be individually arranged as needed. It may include a promoter and / or a terminator. At this time, the ribosome binding sequence may be designed according to the expression level desired to be achieved for each gene.

The transformant in the present invention contains a nucleic acid encoding RNA that enables the induction of gene expression in non-phototrophic C1 metabolizing microorganisms. Such nucleic acids of the invention can be integrated on vectors or chromosomes. If there are two or more nucleic acids to be integrated, all of them may be integrated into the vector, integrated on the chromosome, or some of them may be integrated into the vector and others may be integrated into the chromosome. ..

When the nucleic acid in the present invention is contained in the vector, the vector is not particularly limited, but for example, an origin of replication, a selection marker, or a junction that enables autonomous replication in a non-phototrophic C1 metabolizing microbial cell. It can include a transmission origin. Introduction of the vector into cells can be performed by means known to those of skill in the art. For example, a junction transfer method, an electroporation method, and the like can be mentioned (Kim et al., Applied Biochemistry and Biotechnology, Vol. 73, 81-88 (1998)). The nucleic acid in the present invention can be provided as a vector for carrying out gene modification of a non-phototrophic C1 metabolizing microorganism, and can also be provided as a commercial vector or a kit for gene modification for the same purpose.

It is also a preferred embodiment to integrate the nucleic acid of the present invention on the chromosome of a non-phototrophic C1 metabolizing microorganism which is a host cell. Such incorporation can also be performed by means known to those of skill in the art. For example, Csaki et al. , Microbiology, Vol. The method described in 149,1785-1795 (2003) can be mentioned. The non-phototrophic C1 metabolizing microorganism in which the nucleic acid in the present invention is integrated on the chromosome can also be provided as a commercial host or a kit for gene modification.

The C1 metabolizing microorganism in the present invention refers to a non-phototrophic microorganism capable of metabolizing a compound consisting of 1 carbon, for example, methane, methanol, formaldehyde, formic acid, carbon monoxide, carbon dioxide, methylamine (for example, mono). -, Di-, and tri-methylamine), assimilating bacteria such as methylated thiol, etc., even those that have already been isolated and preserved, those that have been newly isolated from nature, those that have been genetically modified, etc. Any non-C1 metabolizing microorganism such as Escherichia coli or yeast modified so as to be able to metabolize the above compound can be arbitrarily selected. The non-phototrophic C1 metabolizing microorganism in the present invention is preferably a methane-utilizing bacterium or a methanol-utilizing bacterium. These are preferably Methylobacterium, Methylocella, Methanosaeta, Methanosaeta, Burkholderia, Methanosaeta, Methylobacterium, Methylobacterium. Mechirosainasu (Methylosinus), Mechiroshisuchisu (Methylocystis), methylotrophic microbeads um (Methylomicrobium), Methylothermus (Mechirosamasu), Methylomarinum (Mechiromarinamu), Methylovulum (Mechirobarumu), Methanococcus (Methanococcus), Methanothermobacter (methanolate thermo Acetobacter), Metanomonasu (Methanomonas ), Methanosarcina, Metanogenium, Metanobacterium, Metanohalophyllus, Methanosaeta, Methanosaeta, Metanomiclobia, Metanopicrobia Methanosaeta, Methanosaeta, Methanosaeta, Methylobacterium, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas. , Pseudomonas, Pseudomonas, Candida, Saccharomyces, Hansenula, Hansenula, Pichia, Pichia, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas , More preferably, Methanosaeta s capsaturus Bath (NCIMB 11132), Methylophilus methylophilus AS1 (NCIMB 10515), Methylotrophyllus glycogenes (NCIMB 11375) or Paracoccus denitri (NCIMB 11375) or Paracoccus denitris.

The transformant of the present invention can be cultured by a known culture method suitable for the growth of host cells. For example, the medium used in the present invention is a natural medium or a synthetic medium as long as it contains a compound having 1 carbon atom as a carbon source and further contains a nitrogen source, inorganic ions and, if necessary, other organic trace components. Any of them may be used. The culture can be selected from either aerobic or anaerobic, and can be selected according to the purpose such as shaking culture, static culture or aeration stirring culture.

For example, when shaking or statically culturing in a closed system using methane as a carbon source, 1 to 75% of methane gas is added to the gas phase in contact with the medium. When performing aeration culture in an open system, a mixed gas containing 1 to 75% of methane gas is blown into the culture. In any of the above examples, gases such as oxygen, carbon dioxide, and nitrogen can be added as needed. When culturing using methanol as a carbon source, 0.01 to 5% methanol is added to the medium.

As the nitrogen source, potassium nitrate, sodium nitrate, nitric acid, ammonia gas, aqueous ammonia, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate, ammonium phosphate, urea and the like are added to a 0.01 to 10% medium and used. In addition to these, potassium phosphate, sodium phosphate, magnesium sulfate, calcium chloride, iron-EDTA, sodium molybdenate, sodium carbonate, sodium hydrogen carbonate, copper sulfate, copper chloride, iron sulfate, zinc sulfate, manganese chloride are usually used. , Cobalt chloride, nickel chloride, boric acid and other components are added in trace amounts. When the microbial species to be cultured is heterotrophic, 0.01 to 10% of sugars such as glucose and xylose and carbon sources such as glycerol, acetic acid and succinic acid are added.

Induction of expression of a target gene using the riboswitch-based expression induction system of the present invention can be achieved by contacting the transformant as described above with a riboswitch ligand. The ligand can be artificially supplied from the outside or autonomously supplied by the host cell at any time when the expression of the gene is desired to be induced. The latter can be achieved, for example, by selecting metabolites that accumulate in growing cells as ligands (Zhou et al., ACS Synthetic biology, Vol. 4, 1335-1340 (2015)). It is also possible to autonomously supply a ligand by introducing a gene necessary for the biosynthesis of the ligand into the host cell.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples. In the following, unless otherwise specified, the base sequence is described in the direction from the 5'end to the 3'end.

The composition of the medium used in each example is as follows.

(LB medium)
The composition is as follows: 20 g / L LB medium, Lenox (manufactured by Diffco).
[Steam sterilization was performed at 120 ° C. for 20 minutes. If necessary, a final concentration of 50 mg / L kanamycin sulfate and 1.5% Bacto agar (manufactured by Diffco) were added. ]

(NMS medium)
The composition is as follows: 0.8 mM trimethyl 4.7H ₂ O, 10 mM NaNO ₃ , 0.14 mM CaCl ₂ , 1.2 mM NaHCO ₃ , 2.35 mM KH ₂ PO ₄ , _3.4 mM K ₂ HPO ₄ , 20.7 μM Na ₂ MoO _{4.2H 2} O, ₁ μM CuSO 4.5H ₂ O, 10 μM Fe ^III -Na-EDTA, 1 mL trace metal solution (500 mg / L FeSO _{4.7H 2} O, 400 mg / L ZnSO _4.7H ) ₂ O, 20 mg / L MnCl 2.7H ₂ O, 50 mg / L CoCl _{2.6H 2 O} , 10 mg / L NiCl _{2.6H 2} O, 15 mg / L H ₃ BO _{3 ,} 250 mg / L EDTA)
[Phosphate, bicarbonate, CuSO 4.5H ₂ O, Fe ^III _- Na-EDTA, NaHCO ₃ were added after steam sterilization at 120 ° C. for 20 minutes. If necessary, 7.5-10 mg / L kanamycin sulfate, 25 mg / L nalidixic acid, and 1.5% Bacto agar were also added after sterilization. ]

(MM1 medium)
The composition is as follows: 0.2 g / L sulfonyl 4.7H ₂ O, 0.5 g / L (NH4) 2SO4, 2.53 g / L K ₂ HPO ₄ , 2.59 g / L NaH2PO ₄ , ₁ mL trace metal solution (2.2 g / L ZnSO _{4.7H 2} O, 5 g / L Na ₂ EDTA, 733 mg / L CaCl _{2.2H 2 O, 506 mg / L MnCl 2.7H 2 O , 110} mg / L (NH ₄ ) ₆ Mo ₇ O _{24.4H 2} O, 162mg / L CuSO 4.5H ₂ O, 161mg / L CoCl _{2.6H 2} O, _499mg / L FeSO _{4.7H 2} O)
[Steam sterilization was performed at 120 ° C. for 20 minutes. If necessary, 0.5% methanol, 10 mg / L kanamycin sulfate, 25 mg / L nalidixic acid, and 1.5% Bacto agar were also added after sterilization. ]

[Example 1] Induction of sfGFP expression in Escherichia coli (construction of plasmid)
For PCR amplification of nucleic acid fragments required for plasmid construction, Phase polymerase (manufactured by New England Biolabs) was used. An In-Fusion HD Cloning kit (manufactured by Cloning) was used for plasmid construction. Pmk61A was constructed by cloning a hybrid of riboswitch and trc promoter (Ptrc E *) and sfGFP gene into pBHR1 (manufactured by MoBiTech), which is a broad-spectrum host vector. Ptrc E * and sfGFP were synthesized by Genscript. First, the Ptrc E * fragment was amplified by mk316 and mk173R. The sfGFP fragment was then amplified by mk176 and mk317R. Finally, the vector skeleton was amplified by mk318 and mk319R. These three fragments were linked by an In-Fusion HD Cloning kit and E. coli with pmk61A. A clone of colli XL1-Blue (manufactured by Nippon Gene) was obtained. After extracting and purifying the plasmid DNA from the cells, it was confirmed by sequence analysis that pmk61A was correctly obtained. FIG. 1 shows an outline of pmk61A. The above sequence is shown below.

(Induction of expression of sfGFP in Escherichia coli transformant introduced with pmk61A)
pmk61A was introduced into Escherichia coli S17-1 strain (manufactured by Biomedal Co., Ltd.) by the calcium ion method to obtain a transformant. The introduction of the plasmid was confirmed by PCR. Transformant colonies were cultured in 3 mL of LB medium containing 50 mg / L kanamycin at 37 ° C. with shaking overnight, and then planted in 3 mL of theophylline containing 0, 0.1, 2 or 5 mM of theophylline. Subculture was carried out at 37 ° C. for 6 hours with shaking. In addition, as a control, the wild strain was cultured in the same medium containing no theophylline. After removing the culture supernatant by centrifugation, the obtained cells were suspended in distilled water and diluted appropriately. The fluorescence intensity (Ex: 488 nm, Em: 530 nm) was measured with a fluorescence plate reader (infinite200Pro, manufactured by Tecan). Fluorescence intensity values were divided by each OD ₆₀₀ and then subtracted from that in the wild strain (blank). The results are shown in FIG.

[Example 2] Induction of sfGFP expression in Methylococcus capsulatus Bath (introduction of pmk61A into Methylococcus capsulatus Bath)
pmk61A was introduced into Methylococcus capsulatus Bath by a transformation method using conjugation transmission reported by Ali et al. (Ali et al., Microbiology, Vol. 152, 2931-2942 (2009)).

First, pmk61A is electroporated by E. coli, which is a donor of conjugation transmission. Introduced to colli S17-1. The transformant was selected by using the growth on LB agar medium containing 50 mg / L kanamycin as an index, and the sequence of the plasmid was confirmed by sequence analysis. Transformant colonies were suspended in LB medium containing 50 mg / L kanamycin and cultured with shaking at 37 ° C. overnight. 500 μL of overnight culture was inoculated into 25 mL of LB medium containing 50 mg / L kanamycin and cultured with shaking at 37 ° C. until OD ₆₀₀ was around 0.5. The cultured cells were collected by centrifugation, washed 3 times with fresh NMS medium, and then suspended in 1 mL of the same medium.

Simultaneously with the culture of S17-1, Methylococcus capsulatus Bath wild strain (NCIMB 11132), which is a receptor for conjugal transmission, was placed in a 130 mL serum bottle containing 10 mL of fresh NMS medium so that OD ₆₀₀ was 0.03. Inoculated. The bottle was sealed with a butyl rubber septum and 60 mL of bottle air was replaced with the same volume of 10% methane / 90% nitrogen mixed gas. The bottles were cultured at 43 ° C. for 18 hours. The cultured cells were collected by centrifugation and suspended in 5 mL of fresh NMS medium.

After mixing so that the product of OD ₆₀₀ and the volume was 1: 5 between S17-1 and Methylococcus capsulatus Bath, the mixture was filtered through a 0.22 μm nitrocellulose membrane (manufactured by Millipore). This was placed on an NMS agar medium containing 0.02% proteopeptone (manufactured by Diffco) and incubated at 37 ° C. for 24 hours under a 5% methane atmosphere. The cells on the membrane were rinsed with 10 mL of fresh NMS medium, and the cells were collected by centrifugation and then resuspended in 100 μL of the same medium. This was applied to an NMS agar medium containing 7.5 mg / L canamycin and 25 mg / L naridixic acid and incubated under a 5% methane atmosphere at 43 ° C. until colonies appeared. The obtained clone confirmed the presence of the plasmid by PCR.

(Induction of sfGFP expression in Methylococcus capsulatus Bath transformant introduced with pmk61A)
Transformant colonies were inoculated into 130 mL serum bottles containing 10 mL of NMS medium supplemented with 10 mg / L kanamycin and theophylline at any concentration of 0, 0.1, 2, 5, 7.5 or 10 mM. .. In addition, as a control, the wild strain was inoculated into the same medium containing no theophylline. The bottle was sealed with a butyl rubber septum and 60 mL of bottle air was replaced with the same volume of 10% methane / 90% nitrogen mixed gas. Bottles were cultured at 43 ° C. for 24 hours. The culture broth was diluted 10-fold with fresh NMS medium, and the fluorescence intensity (Ex: 488 nm, Em: 530 nm) was measured with a fluorescence plate reader (infinite200Pro, manufactured by Tecan). Fluorescence intensity values were divided by each OD ₆₀₀ and then subtracted from that in the wild strain (blank). The results are shown in FIG. In contrast to the experimental results using E. coli (Fig. 2), no fluorescence during non-induction was detected. At the same time, it also showed good expression induction depending on the theophylline concentration, and the fluorescence intensity when 2 mM theophylline was added was similar to that in Escherichia coli (Fig. 2).

[Example 3] Induction of sfGFP expression in Methylophilus methylophilus AS1 (introduction of pmk61A into Methylophilus methylophilus AS1)
pmk61A was introduced into Methylophilus methylophilus AS1 (NCIMB 10515) by the transformation method using mating transmission reported by Kim et al. (Kim et al., Applied Biochemistry and Biotech 19) Biotech and Biotech. ).

First, pmk61A is electroporated by E. coli, which is a donor of conjugation transmission. Introduced to colli S17-1. The transformant was selected by using the growth on LB agar medium containing 50 mg / L kanamycin as an index, and the sequence of the plasmid was confirmed by sequence analysis. Transformant colonies were suspended in 5 mL of LB medium containing 50 mg / L kanamycin and cultured with shaking at 37 ° C. overnight. The cultured cells were collected by centrifugation, washed 3 times with fresh MM1 medium, and suspended in 1 mL of the same medium.

Simultaneously with the culture of S17-1, a wild strain of Methylophilus methylophilus AS1 which is a receptor for conjugation transmission was inoculated into 25 mL of MM1 medium containing fresh 0.5% methanol and cultured at 30 ° C. overnight. The cultured cells were collected by centrifugation and suspended in 5 mL of fresh MM1 medium.

After mixing so that the product of OD ₆₀₀ and the volume was 1: 2 between S17-1 and Methylophilus methylophilus AS1, the mixture was filtered through a 0.22 μm nitrocellulose membrane (manufactured by Millipore). This was placed on LB agar medium and incubated at 37 ° C. for 24 hours. The cells on the membrane were rinsed with 10 mL of fresh MM1 medium, the cells were collected by centrifugation, resuspended in 1 mL of the medium, diluted 10 ² -fold with 10 mg / L kanamycin, and 0.5% methanol. It was applied to the contained MM1 agar medium and incubated at 37 ° C. until colonies appeared. The obtained clone confirmed the presence of the plasmid by PCR.

(Induction of expression of sfGFP in Methylophilus methylophilus AS1 transformant introduced with pmk61A )
Transformant colonies were inoculated into 25 mL of MM1 medium supplemented with 0.5% methanol, 10 mg / L kanamycin and theophylline at either 0 or 2 mM concentration. In addition, as a control, the wild strain was inoculated into the same medium containing no theophylline. After culturing at 30 ° C. for 24 hours, the culture solution was diluted 10-fold with fresh MM1 medium, and the fluorescence intensity (Ex: 488 nm, Em: 530 nm) was measured with a fluorescence plate reader (infinite200Pro, manufactured by Tecan). Fluorescence intensity values were divided by each OD ₆₀₀ and then subtracted from the autofluorescence value of the wild strain. The results are shown in FIG. The expression intensity when the expression was induced with 2 mM theophylline was 21 times that at the time of non-induction.

[Example 4] Induction of sfGFP expression in Methelylobacillus glycogenes (introduction of pmk61A into Methylovacillus glycogenes )
pmk61A was introduced into Metylobacillus glycogenes (NCIMB 11375) by the transformation method using conjugation transmission reported by Kim et al. (Kim et al., Applied Biochemistry and Biotechnol. ..

First, pmk61A is electroporated by E. coli, which is a donor of conjugation transmission. Introduced to colli S17-1. The transformant was selected by using the growth on LB agar medium containing 50 mg / L kanamycin as an index, and the sequence of the plasmid was confirmed by sequence analysis. Transformant colonies were suspended in 5 mL of LB medium containing 50 mg / L kanamycin and cultured with shaking at 37 ° C. overnight. The cultured cells were collected by centrifugation, washed 3 times with fresh MM1 medium, and suspended in 1 mL of the same medium.

Simultaneously with the culture of S17-1, a wild strain of Metylobacillus gycogenes , which is a receptor for conjugation transmission, was inoculated into 25 mL of fresh MM1 medium containing 1% methanol and cultured at 30 ° C. overnight. The cultured cells were collected by centrifugation and suspended in 5 mL of fresh MM1 medium.

After mixing so that the product of OD ₆₀₀ and the volume was 1: 2 between S17-1 and Methylobacillus glycogenes , the mixture was filtered through a 0.22 μm nitrocellulose membrane (manufactured by Millipore). This was placed on LB agar medium and incubated at 30 ° C. for 24 hours. The cells on the membrane were rinsed with 10 mL of fresh MM1 medium, the cells were collected by centrifugation, resuspended in 1 mL of the medium, diluted 105 ^- fold with 10 mg / L kanamycin, and 0.5% methanol. It was applied to the contained MM1 agar medium and incubated at 30 ° C. until colonies appeared. The obtained clone confirmed the presence of the plasmid by PCR.

(Induction of expression of sfGFP in a methylobacillus glycogenes transformant introduced with pmk61A)
Transformant colonies were inoculated into 25 mL of MM1 medium supplemented with 1% methanol, 10 mg / L kanamycin and theophylline at either 0 or 2 mM concentration. In addition, as a control, the wild strain was inoculated into the same medium containing no theophylline. After culturing at 30 ° C. for 24 hours, the culture solution was diluted 10-fold with fresh MM1 medium, and the fluorescence intensity (Ex: 488 nm, Em: 530 nm) was measured with a fluorescence plate reader (infinite200Pro, manufactured by Tecan). Fluorescence intensity values were divided by each OD ₆₀₀ and then subtracted from the autofluorescence value of the wild strain. The results are shown in FIG. The expression intensity when the expression was induced with 2 mM theophylline was 45 times that at the time of non-induction.

[Example 5] Induction of sfGFP expression in Paracoccus denitrificans (introduction of pmk61A into Paracoccus denitrificans )
pmk61A was introduced into Paracoccus denitrificans (NCIMB 11627) by the electroporation method (Kim et al., Applied Biochemistry and Biotechnology, Vol. 73, 81-88).

Paracoccus denitrificans wild strains were inoculated into 25 mL of fresh MM1 medium containing 1% methanol and cultured overnight at 30 ° C. The cultured cells were collected by centrifugation and suspended in 5 mL of fresh MM1 medium. Incubation was carried out at 30 ° C. until colonies appeared. The obtained clone confirmed the presence of the plasmid by PCR.

(Induction of sfGFP expression in Paracoccus denitrificans transformant introduced with pmk61A)
Transformant colonies were inoculated into 5 mL of LB medium supplemented with 10 mg / L kanamycin and theophylline at either 0 or 2 mM concentration. In addition, as a control, the wild strain was inoculated into the same medium containing no theophylline. After culturing at 30 ° C. for 3 hours, the cells were collected by centrifugation and suspended in distilled water. The fluorescence intensity (Ex: 488 nm, Em: 530 nm) was measured with a fluorescence plate reader (infinite200Pro, manufactured by Tecan). Fluorescence intensity values were divided by each OD ₆₀₀ and then subtracted from the autofluorescence value of the wild strain. The results are shown in FIG. The fluorescence intensity at the time of non-induction was small, and the expression intensity when the expression was induced with 2 mM theophylline was 15 times that at the time of non-induction.

[Example 6] and [Comparative Example 1] Introduction of a lysine decarboxylase gene expression vector into Methylococcus plasmidus Bath (construction of a plasmid)
Pmk96 and pmk99 were constructed by cloning the cadA gene encoding lysine decarboxylase into pmk61A. Based on the report of Kou et al., CadA derived from Alivivrio salmonicida was synthesized by Genscript (Kou et al., Journal of Molecular Catalyst B: Enzymatic, Vol. 133, 88-94).

For the construction of pmk96, the cadA gene region was PCR amplified by mk412 and mk413R. Next, the vector skeleton excluding the riboswitch region was amplified by mk414 and mk415R. These fragments were linked by an In-Fusion HD Cloning kit and E. coli with pmk96. A clone of colli XL1-Blue was obtained. After extracting the plasmid DNA by a conventional method, it was confirmed by sequence analysis that pmk96 was correctly obtained.

For the construction of pmk99, the cadA gene region was PCR amplified with mk425 and mk426R, and the vector backbone was PCR amplified with mk427 and mk428R. These fragments were concatenated with an In-Fusion HD Cloning kit and E. coli with pmk99. A clone of colli XL1-Blue was obtained. After extracting the plasmid DNA by a conventional method, it was confirmed by sequence analysis that pmk99 was correctly obtained. FIG. 7 shows an outline of pmk96 and pmk99. The above sequence is shown below.

(Introduction of pmk96 and pmk99 to Methylococcus capsulatus Bath)
First, pmk96 and pmk99 are electroporated by E. coli, which is a donor of conjugation transmission. Introduced to colli S17-1. The transformant was selected by using the growth on LB agar medium containing 50 mg / L kanamycin as an index, and the sequence of the plasmid was confirmed by sequence analysis. Transformant colonies were suspended in LB medium containing 50 mg / L kanamycin and cultured with shaking at 37 ° C. overnight. 500 μL of overnight culture was inoculated into 25 mL of LB medium containing 50 mg / L kanamycin and cultured with shaking at 37 ° C. until OD ₆₀₀ was around 0.5. The cultured cells were collected by centrifugation, washed 3 times with fresh NMS medium, and then suspended in 1 mL of the same medium.

Simultaneously with the culture of S17-1, Methylococcus capsulatus Bath wild strain (NCIMB 11132), which is a receptor for conjugal transmission, was placed in a 130 mL serum bottle containing 10 mL of fresh NMS medium so that OD ₆₀₀ was 0.03. Inoculated. The bottle was sealed with a butyl rubber septum and 60 mL of bottle air was replaced with the same volume of 10% methane / 90% nitrogen mixed gas. The bottles were cultured at 43 ° C. for 18 hours. The cultured cells were collected by centrifugation and suspended in 5 mL of fresh NMS medium.

After mixing so that the product of OD ₆₀₀ and the volume was 1: 5 between S17-1 and Methylococcus capsulatus Bath, the mixture was filtered through a 0.22 μm nitrocellulose membrane (manufactured by Millipore). This was placed on an NMS agar medium containing 0.02% proteopeptone (manufactured by Diffco) and incubated at 37 ° C. for 24 hours under a 5% methane atmosphere. The cells on the membrane were rinsed with 10 mL of fresh NMS medium, and the cells were collected by centrifugation and then resuspended in 100 μL of the same medium. This was applied to NMS agar medium containing 7.5 mg / L kanamycin and 25 mg / L nalidixic acid, and incubated under a 5% methane atmosphere at 43 ° C. until colonies appeared. When colonies were obtained, the presence of the plasmid was confirmed by PCR.

As a result of the transformation, no transformant of pmk96 was obtained, while a large number of colonies of the transformant of pmk99 were obtained. This result means that the expression of the lysine decarboxylase gene, which competes with the growth of non-phototrophic C1 metabolizing microorganisms, was suppressed to an infinitely small amount by the expression control system of the present invention.

[Example 7] and [Comparative Example 2] Introduction of a lysine decarboxylase gene expression vector into Methylophilus methylophilus AS1 First, pmk96 and pmk99 were conjugated and transmitted by electroporation. Introduced to colli S17-1. The transformant was selected by using the growth on LB agar medium containing 50 mg / L kanamycin as an index, and the sequence of the plasmid was confirmed by sequence analysis. Transformant colonies were suspended in 5 mL of LB medium containing 50 mg / L kanamycin and cultured with shaking at 37 ° C. overnight. The cultured cells were collected by centrifugation, washed 3 times with fresh MM1 medium, and suspended in 1 mL of the same medium.

Simultaneously with the culture of S17-1, a wild strain of Methylophilus methylophilus AS1 which is a receptor for conjugation transmission was inoculated into 25 mL of MM1 medium containing fresh 0.5% methanol and cultured at 30 ° C. overnight. The cultured cells were collected by centrifugation and suspended in 5 mL of fresh MM1 medium.

After mixing so that the product of OD ₆₀₀ and the volume was 1: 2 between S17-1 and Methylophilus methylophilus AS1, the mixture was filtered through a 0.22 μm nitrocellulose membrane (manufactured by Millipore). This was placed on LB agar medium and incubated at 37 ° C. for 24 hours. The cells on the membrane were rinsed with 10 mL of fresh MM1 medium, the cells were collected by centrifugation, resuspended in 1 mL of the medium, diluted 10 ² -fold with 10 mg / L kanamycin, and 0.5% methanol. It was applied to the contained MM1 agar medium and incubated at 37 ° C. until colonies appeared. When colonies were obtained, the presence of the plasmid was confirmed by PCR.

As a result of the transformation, no transformant of pmk96 was obtained, while a large number of colonies of the transformant of pmk99 were obtained. This result also means that the expression of the lysine decarboxylase gene, which competes with the growth of non-phototrophic C1 metabolizing microorganisms, was suppressed to an infinitely small amount by the expression control system of the present invention.

(Plastic deposit)
The plasmid pmk61A described herein is designated as deposit number NITE P-02625, the plasmid pmk96 is designated as deposit number NITE P-02626, and the plasmid pmk99 is designated as deposit number NITE P-02627. Deposited on February 1, 2018.

The nucleic acid of the present invention can be used in non-phototrophic C1 metabolizing microorganisms for gene expression limited to desired conditions, flexible regulation of gene expression level, maximization of substance production performance, and elucidation of the physiological properties of genes through these. It can be used in the industry related to genetic engineering.

Claims (16)

A nucleic acid for controlling gene expression in a non-phototrophic C1 metabolizing microorganism, which is a transformant of a non-photonourishing C1 metabolizing microorganism containing the nucleic acid, which comprises a sequence encoding a riboswitch. The non-phototrophic C1 metabolizing microorganism is selected from the group consisting of Methylococcus capsulatus Bath, Methylophilus methylophilus AS1, Methylophilus glycogenes and Paracoccus zycogenes and Paracoccus denitrificans . Transformant. The transformant according to claim 1 , wherein the theophylline-binding riboswitch has a sequence of 5'-GGTACCGGGTGATACGACATCCGTCTTGATGCCTTGCGCAGCACCCTGCTAAGGAGGCAACAAG-3'(SEQ ID NO: 1). The transformant according to any one of claims 1 or 2 , wherein the riboswitch is located downstream of a natural or artificial promoter. The transformant according to claim 3 , wherein the promoter is an inducible promoter. The transformant according to claim 3 , wherein the promoter is a homeostatic promoter. The transformant according to claim 4 or 5 , wherein the promoter is a trc promoter. The transformant according to any one of claims 1 to 6 , wherein the nucleic acid comprises a natural or artificial ribosome binding sequence. The transformant according to claim 7 , wherein the ribosome-binding sequence has a sequence of 5'-AAGGAGG-3'. The transformant according to claim 7 or 8 , wherein the ribosome-binding sequence is located downstream of the riboswitch via a spacer. The transformant according to any one of claims 1 to 9 , wherein the nucleic acid further comprises a gene to be expressed, and the gene is located upstream of the terminator. The transformant according to any one of claims 1 to 10 , wherein the nucleic acid is integrated on the chromosome of a host microorganism. The transformant according to any one of claims 1 to 10 , wherein the nucleic acid is introduced into a host microorganism as a vector containing the nucleic acid. Non-phototrophic C1 metabolizing microorganisms are selected from Methylococcus transformations Bath (NCIMB 11132), Methylophilus methylophilus AS1 (NCIMB 10515), Methylococcus glycogenes (NCIMBcgenes) (NCIMBcgenes). , The transformant according to any one of claims 1 to 12 . A method for inducing expression of a gene under the control of a riboswitch by supplying a ligand of the riboswitch to the transformant according to any one of claims 1 to 13 . 14. The method of claim 14 , wherein the ligand is theophylline. The method according to claim 14 or 15 , wherein the ligand is autonomously supplied by a transformant.

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