JP2005261361A - Yeast variant and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a yeast variant capable of mass-producing S-adenosylmethionine effective as a remedy of various kinds of diseases for treating liver disease/depression, osteoarthropathy/cancer, or the like; and to provide a method for utilizing the variant. <P>SOLUTION: The yeast variant has a mutation in a gene encoding a methionine-metabolizing enzyme, especially encoding an S-adenosyl-L-homocysteine hydrolase. As a result, abnormality is caused in the methionine-metabolizing system, and a large amount of the S-adenosylmethionine is accumulated in the cell. The S-adenosylmethionine can be mass-produced by culturing the yeast variant. The variant stops the cell cycle at G1 stage at 37°C. As a result, the yeast variant can be utilized for the analysis of in vivo function of the S-adenosylmethionine. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a yeast mutant and use thereof, and more particularly to a yeast mutant capable of accumulating S-adenosylmethionine in a cell and use thereof.

  In recent years, alcoholic liver disease (ALD), which manifests as symptoms such as fatty liver, fibrosis, cirrhosis, and hepatocellular tumor, is a major cause of illness and death worldwide. At present, the relationship between such alcoholic liver disease and S-adenosylmethionine (hereinafter referred to as AdoMet) is drawing attention.

  AdoMet is a physiological compound that exists throughout living tissues and participates in numerous biological reactions as a methyl group donor or enzyme activator in the synthesis and metabolism of hormones, neurotransmitters, phospholipids, and proteins. is there. AdoMet is metabolized by three metabolic pathways: methyl group transfer, sulfur group transfer, and aminopropyl group transfer.

  Hereinafter, the relationship between AdoMet and liver diseases and other diseases will be described.

  First, the relationship between AdoMet and liver disease will be described. AdoMet has been found to have a therapeutic effect on various liver diseases. For example, Non-Patent Document 1 describes that in research using baboons, liver damage caused by ethanol was alleviated by administration of AdoMet. Further, Non-Patent Document 2 describes that administration of AdoMet significantly reduced the mortality rate of cirrhosis patients (persons). Non-Patent Document 3 describes that administration of AdoMet alleviates rat liver damage caused by hepatotoxins such as carbon tetrachloride and acetaminophen.

  AdoMet is known to be involved in the generation of various brain neurotransmitters. Therefore, it has a superior therapeutic effect in the treatment of depression and the like. For example, Non-Patent Document 4 describes the effect of administration of AdoMet on depression patients. For depressed patients, the effect of parenteral and oral administration of 200-1600 mg / d AdoMet was significantly better than the placebo of conventional antidepressants, similar to tricyclic antidepressants. Furthermore, administration of AdoMet synergistically enhances the effects of tricyclic antidepressants, with a faster time to begin to work than conventional antidepressants. In addition, AdoMet has little decrease in therapeutic effect and few side effects in long-term use. However, several cases of mania have been reported in patients with manic depression.

  AdoMet also has a significant therapeutic effect on osteoarthritis. For example, Non-Patent Document 5 describes the results of a comparative study of the therapeutic effects of AdoMet on osteoarthritis with placebo and non-steroidal anti-inflammatory agents in terms of pain relief, functional recovery, and side effects. As a result, AdoMet had the same therapeutic effect as non-steroidal anti-inflammatory agents in reducing pain and restoring function. AdoMet also did not have the side effects often seen with non-steroidal anti-inflammatory drugs.

  In addition, AdoMet is known to be involved in protection against hypomethylation in cells. In cancer cells, a hypomethylated site and a hypermethylated site of DNA on the chromosome are universally seen. This hypomethylated site on the chromosome is regarded as a hallmark of cancer cells. That is, an increase in AdoMet concentration in the cell stimulates the reaction of DNA methyltransferase. This DNA methyltransferase is considered to protect the chromosome from hypomethylation by hypermethylating DNA on the chromosome.

  For example, Non-Patent Document 5 describes the results of a demethylation activity test. In the demethylation activity test, the CMV-GFP plasmid is introduced into HEK293 cells to detect demethylation of this plasmid. The expression of CMV-GFP on the CMV-GFP plasmid is performed by demethylation of the CMV-GFP. Therefore, in Non-Patent Document 5 described above, intracellular demethylation is detected using CMV-GFP expression in NEK293 cells as an index in the presence of AdoMet or adenosylhomocysteine. In addition, in the presence of AdoMet or adenosylhomocysteine, the influence on demethylase (Methylated DNA binding protein 2 / DNA demethylase: MBD2 / dMTase) is also examined in a test tube. As a result, it was shown that adenosylhomocysteine has no inhibitory effect on demethylase, whereas AdoMet directly inhibits the demethylase reaction. The above demethylation test also showed that AdoMet inhibits demethylation in cells. Therefore, AdoMet has been shown to directly inhibit the demethylase reaction in cells resulting in DNA hypermethylation.

  Patent Document 1 discloses amino sugars, glucosaminoglycans, and S- as compositions that can promote anti-inflammatory action, cartilage protection action, cartilage regulation action, cartilage stabilization action, and cartilage metabolism action. A composition comprising adenosylmethionine is disclosed.

  AdoMet becomes S-adenosylhomocysteine (hereinafter referred to as AdoHcy) after donating a methyl group. AdoHcy is hydrolyzed into adenosine and homocysteine by AdoHcy hydrolase.

  The gene encoding the AdoHcy hydrolase has been isolated from various biological species and is very well conserved across species. For example, Patent Document 2 discloses an AdoHcy hydrolase gene isolated from dendritic cells. Patent Document 3 discloses a living organism in which expression of an AdoHcy hydrolase gene is suppressed.

As described above, AdoMet has been found to have a therapeutic effect on various diseases. Therefore, mass production of AdoMet is expected. As such a method for producing AdoMet, for example, Patent Documents 4 and 5 disclose a method for producing AdoMet using yeast.
Lieber CS et al., Hepatology 1990 Feb; 111: 65-72 Lieber CS, Annu Rev Nutr. 2000; 20: 395-430 Gasso M et al., J Hepatol. 1996 Aug; 25: 200-205 Soeken KL et al. J Fam. Pract 2002 May, 51: 425-430 Detich N et. Al. J. Biol. Chem 2003 Jun 6. 20812-20820 Special table 2002-516866 gazette (publication date June 11, 2002) Special Table 2002-513276 Publication (Publication Date May 8, 2002) International Publication No. WO96 / 14734 Pamphlet (International Publication Date May 23, 1996) Japanese Examined Patent Publication No. 4-55677 (Notification Date: September 4, 1992) Japanese Examined Patent Publication No. 4-33439 (Public Notice Date April 3, 1992)

  AdoMet is effective as a therapeutic agent for various diseases such as liver disease, depression treatment, osteoarthritis, and cancer treatment as described above. However, an effective production method has not been established at present.

  AdoMet is also involved in numerous biological reactions as a methyl group donor or enzyme activator in the synthesis and metabolism of hormones, neurotransmitters, phospholipids, and proteins, resulting in abnormalities in the production of AdoMet Isolation of the body provides a model that can be used to elucidate the association between AdoMet and molecular mechanisms in vivo, in particular the association between AdoMet and cell proliferation (cell cycle). Mutants with abnormalities in the production of AdoMet have been isolated mainly from multicellular organisms such as humans, mice and tobacco. However, since these mutants are multicellular organisms, the growth time is extremely long. Therefore, much effort is required to elucidate the relationship between AdoMet and cell proliferation (cell cycle).

  In Patent Documents 4 and 5, a mutant having an abnormality in the production of AdoMet has been isolated from a yeast that can be easily analyzed in molecular biology and has a short growth time. The specific phenotype of is not disclosed. Therefore, even in yeast, the relationship between AdoMet and cell proliferation (cell cycle) has not been elucidated.

  For this reason, elucidation of the phenotype of a yeast mutant having an abnormality in the production of AdoMet is extremely useful as a tool for elucidating the role of AdoMet in cells. In addition, there is a possibility that it can be effectively used for pathological analysis of various diseases related to accumulation of AdoMet, development of therapeutic agents, and improvement of treatment.

  The present invention has been made in view of the above-described conventional problems, and an object thereof is to provide a yeast mutant capable of analyzing the function of AdoMet in vivo and capable of mass-producing AdoMet and use thereof. There is.

  As a result of intensive studies in view of the above problems, the present inventors have found a yeast mutant capable of accumulating AdoMet in cells. And it discovered that an analysis of the function in vivo of AdoMet and mass production of AdoMet could be implement | achieved using this yeast variant, and came to complete this invention. That is, the present invention includes the following inventions.

  (1) A yeast mutant that is capable of accumulating S-adenosylmethionine in a cell, wherein a methionine metabolic enzyme is mutated.

  (2) The methionine metabolic enzyme is S-adenosylhomocysteine hydrolase, and one or more amino acid sequences of the wild-type S-adenosylhomocysteine hydrolase shown in SEQ ID NO: 3 The yeast mutant according to (1), which has a mutant S-adenosylhomocysteine hydrolase having an amino acid sequence substituted, deleted, inserted and / or added.

  (3) The yeast mutant according to (2), wherein the amino acid sequence of the mutant S-adenosylhomocysteine hydrolase is the amino acid sequence represented by SEQ ID NO: 4.

  (4) The yeast mutant according to (2) or (3), which has a gene encoding the mutant S-adenosylhomocysteine hydrolase.

  (5) The yeast mutant according to (4), wherein the gene encoding the mutant S-adenosylhomocysteine hydrolase comprises the base sequence represented by SEQ ID NO: 2.

  (6) The yeast mutant according to any one of (1) to (5), wherein the yeast mutant is Sacharomyces cerevisiae FERMP-19715.

  (7) A method for producing S-adenosylmethionine, comprising producing S-adenosylmethionine by culturing the yeast mutant of any one of (1) to (6) above.

  (8) A screening method for S-adenosylmethionine high-producing yeast, characterized in that the phenotype shown in the absence of methionine is judged using as an index the recovery in the presence of methionine.

  (9) The method for screening a yeast producing high S-adenosylmethionine according to (8), wherein the phenotype is temperature sensitive.

  (10) A screening method for a yeast producing high S-adenosylmethionine, which comprises detecting an amino acid sequence of a methionine metabolic enzyme or a mutation in a gene encoding a methionine metabolic enzyme.

  (11) The method for screening S-adenosylmethionine high-producing yeast according to (10), wherein the methionine metabolic enzyme is S-adenosylhomocysteine hydrolase.

  (12) The amino acid sequence of the wild-type S-adenosyl homocysteine hydrolase shown in SEQ ID NO: 3 consists of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added. A mutant S-adenosylhomocysteine hydrolase characterized by the above.

  (13) The mutant S-adenosyl homocystein according to (12), wherein the amino acid sequence of the mutant S-adenosyl homocysteine hydrolase is the amino acid sequence represented by SEQ ID NO: 4 Hydrolase.

  (14) A gene encoding the mutant S-adenosylhomocysteine hydrolase of (12) or (13) above.

  (15) The gene according to (14), wherein the gene encoding the mutant S-adenosylhomocysteine hydrolase comprises the base sequence represented by SEQ ID NO: 2.

  (16) A recombinant expression vector comprising the gene of (14) or (15) above.

  (17) A method for producing a transformant, wherein the gene according to (14) or (15) is introduced into a host cell punctuation mark.

  (18) A transformant obtained by the method for producing a transformant according to (17) above.

  Since the yeast mutant of the present invention accumulates S-adenosylmethionine in the cell, a large amount of AdoMet can be produced by using the yeast mutant of the present invention for the production of S-adenosylmethionine. Is possible. Furthermore, the yeast mutant of the present invention can be used for elucidating the relationship between intracellular AdoMet and cell proliferation (cell cycle).

  An embodiment of the present invention will be described as follows. Note that the present invention is not limited to this.

(1) Yeast variant according to the present invention The yeast variant of the present invention is a yeast capable of accumulating S-adenosylmethionine in cells, and is a yeast variant in which a methionine metabolic enzyme is mutated.

  First, methionine metabolic system and methionine metabolic enzyme in vivo will be described below.

(1-1) Metabolism of methionine and methionine metabolism enzyme in vivo FIG. 1 shows an outline of a methionine metabolism pathway. In vivo, methionine is metabolized by the following reaction.
(A) Conversion to S-adenosylhomocysteine (AdoHcy) by transmethylation of S-adenosylmethionine (AdoMet).
(B) S-adenosylhomocysteine (AdoHcy) is hydrolyzed to adenosine and homocysteine.
(C) From homocysteine to methionine.
(D) Methionine receives an adenosyl group from ATP in vivo and becomes S-adenosylmethionine (AdoMet).

  Methionine is produced by the reaction pathways (a) to (c) above. In addition, S-adenosylmethionine (AdoMet) is generated again by the reaction (d), and methionine is generated through the reactions (a) to (c).

  Moreover, the homocysteine produced | generated by reaction of (b) turns into cysteine through cystathionine.

  Such metabolism of methionine in the living body is carried out by catalysis of various enzymes (methionine metabolic enzymes). For example, S-adenosylhomocysteine hydrolase is an enzyme that catalyzes the hydrolysis reaction (b). Further, methionine synthase or betaine-homocysteine methyltransferase is known as an enzyme that catalyzes the reaction (c). As an enzyme that catalyzes the reaction (d), methionine adenosyltransferase is known.

(1-2) Mutation of methionine metabolic enzyme In the yeast mutant according to the present invention, the methionine metabolic enzyme is mutated. The mutated methionine metabolic enzyme is not particularly limited as long as it is an enzyme involved in the above methionine metabolism, and may be a yeast mutant in which the methionine metabolic enzyme is mutated. Such mutation of the methionine metabolic enzyme occurs as a result of mutation in the gene encoding the enzyme. That is, it can be said that the yeast mutant according to the present invention has a mutant methionine metabolic enzyme because it has a mutant methionine metabolic enzyme gene. The yeast mutant according to the present invention has a mutation in the mutated methionine metabolic enzyme, thereby causing an abnormality in the methionine metabolic system, and as a result, AdoMet, which is one of the methionine metabolites, accumulates in the cell. . As used herein, “mutation” refers to a difference in traits observed between cells of the same origin or a population thereof. That is, a mutation refers to a trait caused by a difference in gene configuration including a point mutation of a gene or a chromosomal abnormality such as translocation / duplication or deletion. Therefore, “yeast mutant” means a yeast cell or a population of yeast cells in which the mutation has occurred. In addition, the yeast mutant is not limited to those in which mutation has occurred in one gene, and includes multiple yeast mutants in which mutation has occurred in a plurality of genes. The mutation may be a mutation that induces mutation such as UV irradiation or ethyl methanesulfonic acid (EMS) treatment, a natural mutation, or a genetic recombination method. Moreover, the influence on the methionine metabolic enzyme activity by such mutation is not particularly limited, and may be decreased, deleted or increased. A protein / gene having a mutation is referred to as “mutant protein / gene”, and a protein / gene having no mutation is referred to as “wild-type protein / gene”. Furthermore, a gene encoding an enzyme protein is referred to as an “enzyme gene”.

  Here, S-adenosylhomocysteine hydrolase will be described as an example of mutation of a methionine metabolic enzyme. Such S-adenosylhomocysteine hydrolase is also known as S-adenosylcyhomoseine hydratase, which catalyzes the hydrolysis of S-adenosylhomocysteine in the methionine metabolic system as described above, and is a precursor of methionine. It is an enzyme involved in the production of certain homocysteine. The amino acid sequence of S-adenosylhomocysteine hydrolase without mutation (hereinafter referred to as wild type S-adenosylhomocysteine hydrolase) is shown in SEQ ID NO: 3. The S-adenosylhomocysteine hydrolase (hereinafter referred to as mutant S-adenosylhomocysteine hydrolase) mutated by the yeast mutant according to the present invention is a wild type S-adenosyl represented by SEQ ID NO: 3. The amino acid sequence of homocysteine hydrolase is not particularly limited as long as it consists of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added.

  The above "one or more amino acids are substituted, deleted, inserted and / or added" means substitution, deletion or insertion by a known mutant protein production method such as site-directed mutagenesis. , And / or a number that can be added (preferably 10 or less, more preferably 7 or less, and even more preferably 5 or less) amino acids are substituted, deleted, inserted, and / or added. .

  In addition, the yeast mutant of the present invention may be a yeast mutant having a dominant mutation or a recessive mutation, but is particularly preferably a recessive mutation. As used herein, “recessive mutation” refers to a mutation in which a polyploid obtained by multiplying a yeast wild body and a yeast mutant has a phenotype of the polyploid that substantially matches that of the yeast wild body. . The “dominant mutation” refers to a mutation in which the polyploid phenotype substantially matches that of the yeast mutant.

  A yeast mutant having such a recessive mutation has a phenotype almost identical to that of a yeast wild body by introducing a wild type allele into the cell. That is, the phenotype of the yeast mutant is complemented by the wild type allele. Therefore, by introducing a genome library having a genome of a yeast wild body into the yeast mutant, it becomes possible to identify the mutant gene and facilitate analysis. The term “multiplication” means that yeasts of different mating types (for example, a type and α type in budding yeast) are joined.

  Examples of mutant S-adenosylhomocysteine hydrolase include mutant S-adenosylhomocysteine possessed by AdoMet high-producing yeast found by the present inventors (hereinafter referred to as “sah1-1”, which will be described in detail later). A hydrolase is mentioned. The nucleotide sequence of the mutant S-adenosyl homocysteine hydrolase gene is shown in SEQ ID NO: 2, and the amino acid sequence of the S-adenosyl homocysteine hydrolase encoded by it is shown in SEQ ID NO: 4. It was a thing. More specifically, the S-adenosylhomocysteine hydrolase gene of sah1-1 is the thymine of the 836th cytosine (c) of the wild-type S-adenosylhomocysteine hydrolase gene (SEQ ID NO: 1). (T) is substituted (point mutation), and as a result, the 279 threonine (Thr) in the amino acid sequence (SEQ ID NO: 3) of wild-type S-adenosylhomocysteine hydrolase is replaced with isoleucine (Ile). It had been. The yeast mutant having such a mutant S-adenosylhomocysteine hydrolase accumulated AdoMet about 40 times that of wild-type yeast in the cell. The above-mentioned sah1-1 is named “Saccharomyces cerevisiae sah1-1 / scz7” and is registered with the National Institute of Advanced Industrial Science and Technology Patent Biological Consignment Center as deposit number: FERMP-19715 (deposit date: March 2004). 10th).

(1-3) Phenotype of yeast mutant according to the present invention The yeast mutant according to the present invention comprises a mutant methionine metabolic enzyme gene (for example, S-adenosylhomocysteine hydrolase gene) and a mutant methionine metabolic system. Since it has an enzyme (for example, S-adenosylhomocysteine hydrolase), an abnormality occurs in the methionine metabolic system. Therefore, no methionine is produced in the cell, and the yeast mutant cannot normally grow in a state where the amount of methionine necessary for growth of wild-type yeast is present. As a result, the yeast mutant has a different phenotype from the wild yeast. However, such yeast mutants recover their phenotype in a state where more methionine is present than the amount normally required by wild-type yeast, ie, in the presence of methionine.

  As used herein, “phenotype” refers to the morphological and physiological properties of an organism. Moreover, the phenotype of a yeast variant means the character which was different from the phenotype of the yeast in which the mutation has not occurred (hereinafter referred to as a yeast wild body). The yeast mutant phenotype is not particularly limited as long as it is a conventionally known yeast mutant phenotype. Examples thereof include ion sensitivity such as temperature sensitivity, calcium sensitivity and sodium sensitivity, and drugs. Sensitivity.

  For example, the above-mentioned sah1-1 showed temperature sensitivity in the absence of methionine. Such a “temperature-sensitive phenotype” means a phenotype showing growth different from that of a wild yeast body only in a limited temperature range. In addition, the temperature sensitive phenotype includes a high temperature sensitive phenotype that exhibits growth different from that of the wild yeast at a certain temperature or higher, and a low temperature sensitive phenotype that exhibits growth different from that of the wild yeast at a certain temperature or lower. The temperature range is not particularly limited as long as the temperature range is narrower than the temperature range in which the wild yeast can grow (hereinafter referred to as the allowable temperature). For example, when the yeast is a budding yeast Since the allowable temperature is 14 to 38 ° C, the limited temperature range is a temperature range narrower than 14 to 38 ° C. More specifically, temperature sensitive phenotypes include phenotypes such as “cannot grow at 37 ° C.” and “cannot grow at 14 ° C.”.

  In a yeast mutant exhibiting such a temperature-sensitive phenotype, a specific protein or RNA that is the product of the mutated gene becomes unstable at a certain temperature range and loses its original function. As a result, this yeast mutant exhibits a temperature sensitive phenotype. Therefore, experimentally, yeast mutants showing these temperature-sensitive phenotypes can be examined over time for changes in the phenotype from the wild type to the mutant type by changing only the culture temperature. It is effective for functional analysis of genes. Such yeast mutants include yeast mutants whose phenotype is that the temperature sensitive phenotype cannot grow at 14 ° C. and / or 37 ° C.

  The term “in the absence of methionine” as used herein refers to a state in which an amount of methionine necessary for normal growth of wild yeast is present. Therefore, the above “in the presence of methionine” means a state in which a larger amount of methionine required for normal growth of wild yeast is present. In addition, as a quantity of methionine which said yeast wild body requires for normal growth, specifically, the density | concentration of methionine in a culture medium is 0.14 mM.

  The phrase “phenotype is restored” means that the abnormal growth of the yeast mutant is recovered in an environment where the yeast mutant exhibits the phenotype. For example, when the phenotype is a temperature-sensitive phenotype, it means that the limited temperature range is widened and approaches the temperature range in which the wild yeast can grow. More specifically, a yeast mutant exhibiting a temperature-sensitive phenotype “cannot grow at 37 ° C.” can grow at 37 ° C. in the presence of methionine.

  The concentration of the methionine in the medium can be appropriately set according to the phenotype of the yeast mutant. For example, when the phenotype of the yeast mutant is a temperature-sensitive phenotype, the concentration of the methionine in the medium is preferably 1 to 4 mM, and more preferably 2 mM.

  In addition, the above-mentioned `` yeast '' is not particularly limited as long as it is a fungus that passes most of the life cycle in a single cell, budding, or produces a partition wall and divides cells, for example, budding yeast (Saccharocyces cerevisie), Another example is Schizosaccharocyces pombe. In particular, budding yeast (Saccharocyces cerevisie) is preferable.

  On the other hand, the yeast mutant of the present invention may be a suppression mutant of a cell cycle mutant exhibiting an abnormal G2 phase. The “cell cycle” means the periodicity observed in cell division and DNA replication. As a whole, this cell cycle consists of an S phase in which DNA replication is performed, an M phase in which cell division is performed, an interphase G2 phase during transition from S phase to M phase, and G1 during transition from M phase to S phase. Divided into four periods. Therefore, “showing G2 phase abnormality” means that the progression of G2 phase is abnormal in the cell cycle. Specific examples of abnormalities in the G2 phase include “G2 phase progression is delayed”, “G2 phase progression is stopped”, or “G2 phase progression is accelerated”.

  The “cell cycle mutation” means a mutation having a defect in a specific step in the cell cycle in yeast cells. Yeast cells take on different cell morphology at specific steps on the cell cycle described above. Therefore, a cell cycle mutation is also a mutation that makes the shape of most yeast cells uniform under restrictive conditions. The cell cycle mutant means a yeast cell or a population of yeast cells having the cell cycle mutation. Therefore, “a cell cycle mutant exhibiting a G2 phase abnormality” refers to a yeast mutant that is abnormal in the progression of the G2 phase on the cell cycle under restricted conditions.

  Moreover, the phenotype of such a cell cycle variant is not particularly limited as long as it is a phenotype exhibited by a conventionally known cell cycle variant. For example, a temperature sensitive phenotype such as high temperature sensitivity or low temperature, a calcium sensitive phenotype Etc. In particular, the phenotype of the cell cycle mutant is preferably a calcium sensitive phenotype.

  In addition, the “restriction condition” means a condition indicating cell cycle mutation. For example, if the phenotype of the cell cycle variant is a calcium sensitive phenotype, the limiting condition is that in the presence of calcium. Further, when the phenotype of the cell cycle mutant is a high temperature sensitive phenotype incapable of growing at 37 ° C., the restriction condition is 37 ° C.

  In addition, whether such cell cycle mutants exhibit G2 phase abnormalities is determined by comparing the progress of the cell cycle, that is, the time required for each phase of the cell cycle, between the wild yeast and the cell cycle mutant. Can be determined. The method for calculating the progression of the cell cycle is not particularly limited as long as it is a conventionally known method. For example, direct observation of the morphology of yeast cells, introduction of mitotic index, autoradiograph, DNA in each cell by a cell sorter For example, content measurement.

  The yeast mutant according to the present invention includes such suppression mutants of cell cycle mutants.

  The “suppression mutation” refers to a second mutation that is involved in a phenomenon in which the trait represented by the first mutation is canceled. Specifically, the “suppression mutation” refers to a mutation that cancels the G2 phase delay exhibited by the cell cycle mutant. In addition, “suppression mutant” means a yeast cell or a population of yeast cells having the above-described suppression mutation.

  The method for determining whether the mutation of the yeast mutant of the present invention is a suppression mutation of the cell cycle mutant is not particularly limited as long as it is a conventionally known method of determining a suppression mutation. For example, when the cell cycle mutant exhibits a temperature-sensitive phenotype, a double yeast mutant having a cell cycle mutation of the cell cycle mutant and a mutation of the yeast mutant of the present invention is prepared. The phenotype of the double yeast mutant can be determined based on whether the temperature-sensitive phenotype inherent in the cell cycle mutant is eliminated.

  According to recent findings, AdoMet not only functions as an intermediate medium in methyl group donor and methionine metabolism, but also regulates essential liver functions such as liver damage detection and liver regeneration / differentiation. It has been suggested to be a switch (Corrales FJ, J. Nutr. 2002 Aug; 132). However, it is unclear at present what function AdoMet switches in the cell. As described above, the yeast mutant of the present invention is a suppressive mutant that accumulates AdoMet in the cell and has an abnormality in the G2 phase, and therefore, AdoMet and cell proliferation (cell cycle). ) Is extremely useful as a tool for elucidating the relationship. In addition, the yeast mutant of the present invention may be effectively used for pathological analysis of various diseases related to accumulation of AdoMet, development of therapeutic agents, and improvement of treatment.

(1-4) Example of acquisition of yeast mutant according to the present invention In the budding yeast, the present inventors used a ZDS1 gene mutant that causes G2 phase delay in the presence of calcium as the cell cycle mutant. This ZDS1 gene mutant shows an abnormal cell morphology in which the bud extends in the presence of calcium, and the progression of the G2 phase of the cell cycle is delayed. As a suppression mutant of the calcium-sensitive phenotype of this ZDS1 gene mutant, a yeast mutant (zds1Δsah1-1; details will be described later) obtained by mutating the gene encoding S-adenosylhomocysteine hydrolase is obtained. did.

  Furthermore, as shown in Example 5 described later, the present inventor has clarified that AdoMet delays the progression of the G1 phase of the cell cycle by suppressing the transcription of the cell cycle regulator. Thus, in the budding yeast, the present invention is the first to show the relationship between AdoMet and cell proliferation (cell cycle).

  Therefore, this zds1Δsah1-1 is extremely useful as a tool for elucidating the relationship between AdoMet and cell proliferation (cell cycle), and the yeast mutant of the present invention is used for pathological analysis of various diseases related to AdoMet accumulation. There is also a possibility that it can be effectively used for the development and improvement of treatments.

(2) Screening method according to the present invention The screening method according to the present invention is aimed at screening AdoMet high-producing yeast. Here, the “AdoMet high-producing yeast” means that the methionine metabolic enzyme mutated as described above causes an abnormality in the methionine metabolic system, and as a result, the intracellular accumulation amount of AdoMet increased compared to the wild type yeast. It is yeast, that is, the yeast mutant according to the present invention explained in (1) above. The increase amount (rate) of the accumulated amount of AdoMet is not particularly limited as long as it exceeds the accumulated amount of AdoMet of wild-type yeast.

  Specifically, as a method of screening for such AdoMet high-producing yeast, (a) a method of judging, as an index, that a phenotype shown in the absence of methionine, for example, a temperature-sensitive phenotype, is restored in the presence of methionine; b) There is a method for detecting an amino acid sequence of a methionine metabolic enzyme such as S-adenosyl homocysteine hydrolase or a mutation of a gene encoding a methionine metabolic enzyme such as S-adenosyl homocysteine hydrolase.

  First, (a) will be described. As described in (1-3) above, the yeast in which the methionine metabolism abnormality has occurred exhibits various phenotypes (temperature sensitivity, etc.) in the absence of methionine as described in (1-3) above. In the presence of methionine, the phenotype is restored. The screening method according to the present invention is to screen AdoMet high-producing yeast in which a mutation has occurred in a methionine metabolic enzyme by detecting the phenomenon.

  More specifically, it will be as follows. Note that temperature sensitivity will be described as an example of the phenotype. First, yeast that shows temperature sensitivity in the absence of methionine is selected from yeast that has been artificially mutagenized or yeast that has not been mutagenized. Temperature sensitivity is a phenotype that cannot grow at a temperature at which wild-type yeast can grow as described above. Therefore, when screening for budding yeast that can grow at 14 to 38 ° C. in the wild type, selection may be made based on “cannot grow at 37 ° C.”, “cannot grow at 14 ° C.”, or the like. Next, with respect to the yeast exhibiting temperature sensitivity in the absence of methionine, a yeast whose temperature sensitivity is restored in the presence of methionine, that is, “can grow at 37 ° C.” or “can grow at 14 ° C.” is selected. The yeast thus obtained is a yeast in which an abnormality occurs in the methionine metabolic system, and is likely to be a target high-producing AdoMet yeast. The production amount (accumulated amount) of AdoMet in the selected yeast can be measured from a yeast cell extract using detection by a conventionally known paper chromatography or detection by capillary electrophoresis. The artificial mutation treatment method is not particularly limited, and examples thereof include mutagenesis with ethylmethanesulfonic acid (EMS) or ultraviolet (UV) irradiation.

  In addition, as a method for identifying which gene is mutated in the AdoMet high-producing yeast screened in (a) above, a method for obtaining a gene that complements the phenotype of a conventionally known yeast mutant may be used. For example, although not particularly limited, for example, a genomic library that covers the genome of wild-type yeast is introduced into the AdoMet high-producing yeast screened in (a) above, and the phenotype of this AdoMet high-producing yeast is wild. A method for obtaining a clone complemented to substantially the same level as that of the type yeast can be mentioned. Then, by detecting the gene for this clone, it is possible to identify which gene is mutated in the AdoMet high-producing yeast. The method for detecting the gene is not particularly limited, and a known method such as detection by sequencing, detection by restriction enzyme map, detection by Southern blotting, detection by DNA microarray or the like may be used.

  Next, the method (b) will be described. The method involves mutation of an amino acid sequence of a yeast methionine metabolic enzyme (eg, S-adenosylhomocysteine hydrolase) or a gene encoding a methionine metabolic enzyme (eg, S-adenosylhomocysteine hydrolase). This is a method for directly detecting. Either amino acid sequence mutations or gene mutations may be detected. However, operations such as protein purification and amino acid sequencing are complicated, and therefore it is easier to detect gene mutations. The method for detecting the gene mutation is not particularly limited, and a known method such as detection by sequencing, detection by restriction enzyme map, detection by Southern blotting, detection by DNA microarray, etc. may be used.

  For screening AdoMet high-producing yeast, the above method (a) or (b) may be used alone, or (a) and (b) may be used in combination. For example, candidate strains may be narrowed down in advance by the method (a), and substitution of genes or the like may be detected for the candidate strains. By combining the two screening methods in this way, it becomes possible to select the target AdoMet high-producing yeast efficiently and with high probability.

(3) Method for producing AdoMet according to the present invention The method for producing AdoMet according to the present invention is to prepare AdoMet by culturing the above-described yeast mutant (AdoMet high-producing yeast). Since the yeast mutant of the present invention accumulates AdoMet in the cells, mass production of AdoMet can be realized by extracting and purifying AdoMet from the cultured yeast mutant. Below, the production method of the AdoMet will be specifically described.

  Conventionally known yeast culture conditions can be applied to the yeast mutant culture conditions for mass production of AdoMet. For example, it is preferable to carry out under aerobic conditions in a liquid medium containing methionine, a carbon source, a nitrogen source, an inorganic salt, and an organic micronutrient source.

  Methionine may or may not be added as long as the wild yeast is contained in the medium in an amount necessary for normal growth. However, when methionine is added, it is usually 0.02 g / dl. It is preferable to add at the above ratio. The method for adding methionine may be any of the method of adding the whole amount at once, or the method of adding in a divided manner and sequentially. However, when the amount of methionine added is large, the amount of S-adenosylmethionine accumulated in cells tends to decrease when the former method is adopted. In such a case, the latter method is adopted. Is preferred.

  Carbon sources include sugars such as glucose, sucrose, and fructose; alcohols such as ethanol and glycerin; and starch hydrolysates containing these, molasses, soybean whey, fruit juice waste, fish processing waste, fermentation waste, pulp Waste liquids can also be used. As the nitrogen source, urea, ammonium succinate, ammonium citrate, ammonium lactate and the like are preferable. Inorganic salts include phosphates such as calcium phosphate, potassium phosphate, sodium phosphate and lithium phosphate, potassium salts such as potassium chloride, sodium salts such as sodium chloride and sodium carbonate, magnesium salts such as magnesium sulfate and magnesium chloride. Conventionally known inorganic salts such as manganese salts such as manganese sulfate and manganese chloride, iron salts such as iron sulfate and iron chloride, zinc salts, copper salts and cobalt salts can be used as necessary. Organic trace nutrient sources include vitamins, amino acids, yeast extract containing these, meat extract, malt extract, corn steep liquor, casamino acids, soy flour, soy hydrolyzate, peptone, tryptone, casein decomposition solution as required Can be used.

  The culture condition is not particularly limited as long as the culture is performed under an aerobic condition. For example, while controlling the pH of the medium to 3 to 8, preferably 3.5 to 7, By culturing at 45 ° C., preferably 20 ° C. to 35 ° C., more preferably 25 ° C. to 33 ° C. for 2 to 10 days, AdoMet is produced and accumulated in the yeast mutant cells.

  In the method for producing AdoMet of the present invention, after culturing the yeast mutant, the yeast mutant is separated from the medium, and then AdoMet is extracted and purified from the yeast mutant. The method used in these steps is not particularly limited, and a conventionally known method may be used. That is, in the step of separating the yeast mutant from the medium, for example, a method by centrifugation, a method by filtration, and the like can be mentioned. In addition, in the process of obtaining AdoMet from the yeast mutant, AdoMet is extracted with an extractant such as perchloric acid, hydrochloric acid, sulfuric acid, formic acid, acetic acid, formic acid ester, acetic acid ester, and ethanol, and then extracted according to a conventionally known method. By purifying AdoMet in the liquid, high purity stabilized AdoMet is obtained.

  As a method for purifying AdoMet, a conventionally known method can be applied. For example, S-adenosylmethionine is purified by precipitation using activated carbon, strong acid cation exchange resin, weak acid cation exchange resin, chelate resin, chromatographic method, Rheinecke salt, picric acid, phosphotungstic acid, picrolic acid, etc. And a method of extracting AdoMet using an organic solvent such as acetone and ethanol, and the like, and can be performed in combination as appropriate. In this case, in order to stably obtain AdoMet, an acid such as sulfuric acid, paratrienesulfonic acid, sulfosalicylic acid, etc. is generally added and recovered in the form of AdoMet salt or double salt.

  The present invention also includes AdoMet produced by the above-described AdoMet production method. The AdoMet of the present invention can be mass-produced by the above manufacturing method. Therefore, it is possible to provide inexpensive, high purity and stable AdoMet.

(4) Mutant S-adenosylhomocysteine hydrolase and mutant S-adenosylhomocysteine hydrolase gene according to the present invention are shown in SEQ ID NO: 3. In the amino acid sequence of the wild-type S-adenosylhomocysteine hydrolase shown, the mutant is characterized by consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added. S-adenosyl homocysteine hydrolase, for example, a mutant S-adenosyl homocysteine hydrolase having the amino acid sequence shown in SEQ ID NO: 4. On the other hand, the S-adenosylhomocysteine hydrolase gene according to the present invention is characterized in that it comprises the gene encoding the above mutant S-adenosylhomocysteine hydrolase, for example, the base sequence shown in SEQ ID NO: 2. To do.

  The mutant S-adenosylhomocysteine hydrolase and the method for obtaining the mutant S-adenosylhomocysteine hydrolase gene (production method) according to the present invention are not particularly limited. Examples of the following methods can be given.

(4-1) Method for obtaining mutant S-adenosylhomocysteine hydrolase according to the present invention As a method (production method) for obtaining the mutant S-adenosylhomocysteine hydrolase of the present invention, A simple purification method from cells, tissues, etc. expressing the mutant S-adenosylhomocysteine hydrolase of the invention can be mentioned. The purification method is not particularly limited, and a cell extract may be prepared from cells or tissues by a known method, and the cell extract may be purified using a known method such as a column.

  Moreover, as a method for obtaining the mutant S-adenosylhomocysteine hydrolase of the present invention, a method using a gene recombination technique or the like can also be mentioned. In this case, for example, the mutant S-adenosylhomocysteine hydrolyzed above obtained by incorporating the mutant gene of the present invention into a vector or the like, introducing it into a host cell so that it can be expressed by a known method, and translating in the cell. For example, a method of purifying a degrading enzyme can be employed.

  When introducing a foreign gene into the host in this way, there are various expression vectors and hosts that incorporate a promoter that functions in the host for the expression of the foreign gene. do it. The method for purifying the produced protein varies depending on the host used and the nature of the protein, but the target protein can be purified relatively easily by using a tag or the like.

  The method for producing the mutant protein is not particularly limited. For example, site-directed mutagenesis (Hashimoto-Gotoh, Gene 152, 271-275 (1995), etc.), PCR method for introducing point mutations into base sequences and creating mutant proteins, or transposon insertion A well-known mutant protein production method such as a mutant strain production method can be used. A commercially available kit may be used for the production of the mutant protein.

  The method for obtaining the mutant S-adenosylhomocysteine hydrolase according to the present invention is not limited to the method described above, and may be, for example, chemically synthesized. Alternatively, the mutant protein of the present invention may be synthesized from the mutant gene of the present invention using a cell-free protein synthesis solution.

(4-2) Method for obtaining mutant S-adenosylhomocysteine hydrolase gene according to the present invention The method for obtaining a mutant S-adenosylhomocysteine hydrolase gene according to the present invention is particularly limited. Although it is not a thing, the acquisition method using the yeast variant concerning this invention mentioned above is mentioned, for example. More specifically, for example, an acquisition method by a PCR method can be mentioned. In this method, first, primers are designed from the 5 ′ side and 3 ′ side sequences (or their complementary sequences) from the nucleotide sequence information of the wild-type S-adenosyl homocysteine hydrolase gene. Next, PCR is performed using these primers as a template with genomic DNA (or cDNA), etc., and a DNA region sandwiched between the two primers is amplified, so that a large amount of the DNA fragment containing the mutant gene of the present invention can be obtained. Can be acquired. By sequencing the mutant gene obtained at this time, it is possible to easily identify where the mutation occurs in the mutant gene of the present invention, that is, the mutation point of the mutant gene of the present invention. it can.

(5) Recombinant expression vector according to the present invention, method for producing transformant according to the present invention, and transformant (5-1) Recombinant expression vector according to the present invention It contains a mutant S-adenosylhomocysteine hydrolase gene. For example, a recombinant expression vector into which cDNA is inserted can be mentioned. For the production of the recombinant expression vector, a plasmid, phage, cosmid or the like can be used, but it is not particularly limited. In addition, a manufacturing method may be performed using a known method.

  The specific type of vector is not particularly limited, and a vector that can be expressed in a host cell may be appropriately selected. That is, according to the type of host cell, an appropriate promoter sequence is selected to ensure gene expression, and the mutant S-adenosylhomocysteine hydrolase gene according to the present invention is incorporated into various plasmids and the like. May be used as an expression vector. Moreover, DNA segments other than promoters, such as a terminator, may be included.

(5-2) Method for producing transformant according to the present invention The method for producing a transformant according to the present invention is characterized in that the mutant S-adenosylhomocysteine hydrolase gene is introduced into a host. . The method for introducing the gene into the host cell is not particularly limited, and the gene may be introduced by a known method using the recombinant vector of the present invention. As a known gene introduction method, that is, a transformation method, an electroporation method, a calcium phosphate method, a liposome method, a DEAE dextran method, a lithium acetate method, or the like can be suitably used.

  The host cell is not particularly limited, and various conventionally known cells can be suitably used. Specifically, for example, insects such as Drosophila melanogaster, bacteria such as Escherichia coli, yeast (budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe), nematode Caenorhabditis elegans, Xenopus laevis oocytes Etc. In addition, if a promoter or vector is selected, plants can be targeted for transformation. Among them, the above-described yeast is suitable as the target organism. Yeast can be easily analyzed for genetics, and a transformant can be produced in a short time. Therefore, it becomes easier to elucidate the relationship between intracellular AdoMet and cell proliferation (cell cycle). Furthermore, the mutant S-adenosylhomocysteine hydrolase gene may be introduced into a host cell (yeast) from which the S-adenosylhomocysteine hydrolase gene has been deleted. By doing so, only the mutant S-adenosylhomocysteine hydrolase gene of the present invention can be expressed, and an AdoMet high-producing transformant (yeast) can be obtained.

  In addition, in order to confirm whether or not the mutant S-adenosylhomocysteine hydrolase gene according to the present invention has been introduced into the host cell, and whether or not it is reliably expressed in the host cell, various markers are used. May be used. For example, a gene deleted in the host cell is used as a marker, and a plasmid containing this marker and the mutant gene of the present invention is introduced into the host cell as an expression vector. Thereby, the introduction of the mutant gene of the present invention can be confirmed from the expression of the marker gene. Alternatively, the mutant S-adenosylhomocysteine hydrolase according to the present invention may be expressed as a fusion protein. For example, the green fluorescence protein GFP (Green Fluorescent Protein) derived from Aequorea jellyfish may be used as a marker, and the protein according to the present invention may be expressed as a GFP fusion protein.

(5-3) Transformant According to the Present Invention The transformant according to the present invention is a transformant obtained by the method for producing a transformant according to the present invention. That is, it is a transformant in which the mutant S-adenosylhomocysteine hydrolase gene according to the present invention is introduced. Here, “gene introduced” means that the gene is introduced into a target cell (host cell) so as to be expressed by a known genetic engineering technique (gene manipulation technique). The “transformant” means not only cells / tissues / organs but also individual organisms. The transformant according to the present invention, particularly a transformed cell obtained by introducing a mutant S-adenosylhomocysteine hydrolase gene into a host cell (yeast) lacking the S-adenosylhomocysteine hydrolase gene ( It is possible to produce AdoMet at a high rate by culturing yeast).

(6) Utilization method (usefulness) of yeast mutants and the like according to the present invention
Any of the yeast mutant according to the present invention, AdoMet production method, AdoMet high production yeast screening method, mutant S-adenosylhomocysteine hydrolase and the gene, recombinant expression vector, transformation method and transformant Can also be used for mass production of AdoMet. It can also be used to elucidate the relationship between intracellular AdoMet and cell proliferation (cell cycle). A method of using AdoMet produced by the present invention will be described below.

  AdoMet can be used as a therapeutic agent for various diseases. For example, examples of the disease include liver disease, depression treatment, osteoarthritis, and cancer treatment.

  As shown in the methionine metabolic pathway described above, the reaction for generating AdoMet from methionine is performed by methionine adenosyltransferase. In mammals, there are three genes encoding methionine adenosyltransferases: MAT1, MAT2, and MAT3. Among these, the MAT1 gene is known to be expressed specifically in the liver. Moreover, it is known that the solid of alcoholic cirrhosis has remarkably reduced activity of the MAT1 protein expressed specifically in the liver. In the MAT1 gene knockout mouse, methionine metabolism disorder occurs, and a decrease in the amount of AdoMet in the liver, liver enlargement, and fatty liver symptoms are observed.

  In addition, non-alcoholic steatohepatitis occurred in MAT1 gene knockout mice. In this knockout mouse, carbon tetrachloride-induced liver toxicity and malignant tumor incidence were examined at 3 months and 18 months of age, respectively. As a result, in the MAT1 gene knockout mice, fat peroxidation was increased and liver damage induced by carbon tetrachloride was promoted. In knockout mice, the development of liver malignant tumors was observed in more than half of 18-month-old mice. These results indicate that AdoMet plays an important role in maintaining normal liver function and suppressing liver tumorigenesis (Martinez-Chantar ML, FASE J 2002 Aug; 16).

  Also, AdoMet prescription has the effect of reducing liver damage caused by various drugs and improving the survival of cirrhosis in alcoholic patients (Avila MA et al. Alcohol 2002 Jul 27).

The most important metabolic pathway for the health of the liver in the body is the two pathways in the above-described methionine metabolic pathway that generate methionine and AdoMet by homocysteine methylation. Ethanol has been suggested to inhibit either of these two pathways catalyzed by methionine synthase. Ethanol itself does not inhibit the activity of the enzyme. Therefore, AdoMet in the liver has been shown to be essential for the transport of fat from the liver to prevent hepatic steatosis and the resulting liver damage. (Barak AJ et. Al. Alcohol Feb: 26)
Thus, AdoMet plays an important role in the treatment of liver diseases such as liver disorders and liver tumors.

  AdoMet is a methyl group donor and is involved in various brain neurotransmitters. In particular, AdoMet depressive activity has attracted attention. Brain polyamines play an important role in neurogenesis. Examples of brain polyamines include putrescine, spermidine, spermine and the like. AdoMet is essential for the synthesis of this brain polyamine. In addition, depression is said to be associated with a partial decrease in brain volume, and depression inhibitors have been shown to increase neurogenesis in certain brain areas and affect neurogenesis .

  Levels of putrescine, spermidine, and spermine, brain polyamines in the hippocampal body, and putrescine in the nucleus accumbens nucleus in the depleted rat induced by moderately applying chronic and unpredictable stress Has been found to be significantly reduced (Gendani S et. Al. Neuroreport 2001 Dec 21). When this experiment rat was given a sufficient amount of AdoMet as a depressing effect, it was shown that the amount of spermidine and spermine in the hippocampal body and the amount of putrescine in the septal nucleus were restored. Yes.

  Thus, AdoMet plays an important role in the treatment of depression.

  AdoMet is effective in reducing pain and restoring function in the treatment of osteoarthritis. Therefore, the therapeutic agent of the present invention can be preferably used for the treatment of such osteoarthritis.

  Embodiments will be described below in more detail with reference to the accompanying drawings. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention.

  In this example, unless otherwise specified, experimental procedures are as follows: (1) Nature 392 303-306 (1998) Mizunuma et al, (2) EMBO J. 20 1074-1085 (2001) Mizunuma et al, (3) Proc Natl. Acad. Sci. USA (2004) In printing The method described in Mizunuma et al was followed. In this example, a ZDS1-deleted yeast mutant (hereinafter referred to as zds1Δ) in which the function of the ZDS1 protein was completely deleted was used as the ZDS1 yeast mutant.

[Example 1]
In this example, acquisition of SAH1 yeast mutant will be described. The SAH1 yeast mutant was obtained as a suppression mutant of the calcium sensitive phenotype of the ZDS1 yeast mutant.

  As shown in FIG. 2 (a), the ZDS1 yeast mutant is unable to grow on a medium containing high concentration calcium (300 mM). Many mutants that can grow even under this calcium concentration condition were obtained. Among these mutants, a yeast mutant (hereinafter zds1Δsah1-1) in which a mutation occurred in the SAH1 gene was obtained.

  As shown in FIG. 2A, this yeast mutant significantly suppresses the calcium sensitivity indicated by zds1Δ. Moreover, not only such a suppression mutation but the high temperature sensitivity phenotype which cannot be grown at 37 degreeC, and the low temperature sensitivity phenotype which cannot be grown at 14 degreeC were shown.

  For identification of the SAH1 gene, a yeast genomic library was introduced into this yeast mutant, and the target plasmid was obtained as a clone complementary to the above phenotype. As a result of sequencing the genome insertion part of this target plasmid and searching the database, it was revealed that this plasmid contains the SAH1 gene. Next, a clone containing only the SAH1 gene in this yeast mutant was introduced with a low copy. As a result, the phenotype of this yeast mutant was complemented by the introduction of a clone containing only the SAH1 gene, so that this yeast mutant was predicted to have a mutation in the SAH1 gene. Hereinafter, the yeast mutant obtained by this screening is referred to as SAH1 yeast mutant (hereinafter referred to as “sah1-1”).

  In order to confirm whether or not the SAH1 gene was actually mutated, the mutation point of the sah1-1 mutant gene in the sah1-1 was identified. Yeast genome was prepared from sah1-1 and sequenced. In addition, the genome of the used yeast wild body was used for control. As a result, sah1-1 was a point mutation in which the 836rd cytosine (c) of the SAH1 gene was replaced with thymine (T). In addition, due to this point mutation, threonine (Thr), which is the 279th amino acid, was changed to isoleucine (Ile) in the amino acid sequence.

  Furthermore, in order to confirm whether the point mutation is necessary for the function of the SAH1 protein, a plasmid in which the point mutation was introduced into the wild-type SAH1 gene was constructed. When this plasmid was introduced into the above-mentioned sah1-1, the phenotype of sah1-1 was not complemented. From these results, it was concluded that the yeast mutant obtained by the above screening was actually a yeast mutant in which a mutation occurred in the SAH1 gene.

  FIG. 2 is a diagram showing the phenotype of the obtained yeast mutant (zds1Δsah1-1, sah1-1). As shown in FIG. 2 (a), zds1Δsah1-1 shows a low temperature sensitive phenotype (cannot grow at 14 ° C.) and a high temperature sensitive phenotype (cannot grow at 37 ° C.). Zds1Δ shows a calcium-sensitive phenotype that cannot grow in the presence of calcium, but when this yeast mutant was mutated in the SAH1 gene (ie, zds1Δsah1-1), it did not show a calcium-sensitive phenotype. Therefore, it can be seen that zds1Δsah1-1 is a suppression mutant of zds1Δ. Sah1-1 also exhibits a low temperature sensitive phenotype (cannot grow at 14 ° C) and a high temperature sensitive phenotype (cannot grow at 37 ° C).

  In addition, as shown in FIG. 2B, zds1Δ shows a cell morphology abnormality in which budding has an elongated cell shape in the presence of calcium. In the state of zds1Δsah1-1, such a cell morphology abnormality Not shown.

  In addition, as shown in FIG. 2C, zds1Δ has a delay in the G2 phase of the cell cycle in the presence of calcium, but in the state of zds1Δsah1-1, this delay in the G2 phase was not observed.

  From the above, it was found that the SAH1 gene mutation suppresses not only the calcium-sensitive phenotype of zds1Δ but also abnormal cell morphology and G2 phase delay.

[Example 2]
In this example, the relationship between sah1-1 and zds1Δ in cell proliferation (cell cycle) was analyzed. The result is shown in FIG.

  First, transcription amounts of cell cycle regulators were measured in wild yeast (hereinafter referred to as wild type), zds1Δ, zds1Δsah1-1, and sah1-1, and compared in the absence of calcium and in the presence of calcium. Specifically, the above-mentioned various yeasts are cultured in the absence of calcium or in the presence of calcium, and the transcription amounts of the SWE1 gene and CLN2 gene, which are cell cycle regulators, in various yeast mutants after 1 hour are Northern Northern. It was investigated by analysis. The result is shown in FIG. As shown in FIG. 3A, zds1Δsah1-1 and sah1-1 suppressed the transcription of the SWE1 gene and the CLN2 gene regardless of the absence of calcium or the presence of calcium.

  Next, the various yeast mutants described above were cultured in the absence of calcium or in the presence of calcium, and the amounts of SWE1 protein and CLN2 protein in the various yeast mutants after 1 hour were examined by Western analysis. The result is shown in FIG. As shown in FIG. 3C, zds1Δsah1-1 and sah1-1 decreased the amounts of SWE1 protein and CLN2 protein in the absence of calcium or in the presence of calcium.

  Next, the transcription of the SWE1 gene and the CLN2 gene on the cell cycle in wild type and zds1Δsah1-1 was analyzed in more detail. Specifically, wild type and sah1-1 were synchronized with the G1 phase of the cell cycle using a peptide called α-factor, then shifted to a medium without α-factor, and the cell cycle was changed at 25 ° C. Proceeded. Sampling was performed every 20 minutes for wild type and sah1-1, which progressed synchronously in this way. And about each sample, while examining DNA content by FACS analysis, the transcription | transfer amount of SWE1 gene and CLN2 gene was investigated by Northern analysis. The result is shown in FIG.

  The right side of FIG. 3 (c) shows the DNA content examined by FACS analysis. The cell cycle progression of the various yeast mutants can be seen by observing this change in DNA content over time. And the left side of FIG.3 (c) shows the transcription | transfer change of SWE1 gene and CLN2 gene on this cell cycle. As shown in the FACS analysis on the right side, in the wild type, the DNA content shifts from 1C to 2C and is in the G2 phase at 20 and 40 minutes after the shift. As shown in the northern analysis result on the left side, transcription of the SWE1 gene and the CLN2 gene appears at 20 minutes and 40 minutes after the shift. Transcription of the SWE1 gene and the CLN2 gene disappears 60 minutes after the shift. Thus, in the wild type, transcription of the SWE1 gene and the CLN2 gene has a specific periodicity on the cell cycle. On the other hand, in Sah1-1, the increase in the SWE1 gene and CLN2 gene observed in wild type was not observed. From the FACS analysis, when the DNA content was compared between wild type and sah1-1, a delay in the G1 phase was observed even at 25 ° C. where sah1-1 grows.

  From the above, it has been clarified that in the case of sah1-1, progression of the G1 phase is delayed by suppressing transcription of a cell cycle regulator.

Example 3
As a result of database search, the above-mentioned SAH1 gene was found to encode S-adenosyl-L-homocysteine hydrolase. If so, AdoHcy, a precursor of homocysteine, is expected to accumulate in the cells of sah1-1. Thus, with respect to yeast wild body and sah1-1 cultured in YPD medium or O medium to the logarithmic growth phase, intracellular AdoMet and AdoHcy were measured using capillary electrophoresis, and wild type and sah1- 1 and compared. The results are shown in Table 1.

  As a result, as shown in Table 1, accumulation of AdoHcy and AdoMet in cells was observed in sah1-1. The accumulated amount was about 8 times with AdoHcy and about 37 times with AdoMet as compared to the wild type (result of O medium).

  From this, it was concluded that sah1-1 is deficient in the activity of S-adenosyl-L-homocysteine (AdoHcy) hydrolase.

Example 4
In Example 3 above, it was revealed that the activity of S-adenosyl-L-homocysteine (AdoHcy) hydrolase is deficient, so it is expected that sah1-1 has a defect in the methionine metabolic pathway. Is done. Therefore, the growth of sah1-1 in the presence of methionine, AdoMet, or AdoHcy, which is a product in the methionine metabolic pathway, was examined. Specifically, growth at 25 ° C. in the presence of methionine, AdoMet, or AdoHcy was compared between wild type and sah1-1. The result is shown in FIG. In addition, “-” described in FIG. 4 means a medium containing no methionine in the medium.

  As shown in FIG. 4, sah1-1 cannot grow on a medium containing no methionine. In contrast, in the presence of methionine or AdoMet, sah1-1 can grow. That is, it was revealed that sah1-1 restores the phenotype that it cannot grow at 25 ° C. in the presence of methionine or AdoMet.

Example 5
In this example, the relationship between AdoMet and cell proliferation (cell cycle) was analyzed. That is, in this example, the influence of the cell cycle progression of wild type in the presence of methionine, AdoMet, or AdoHcy was examined. The result is shown in FIG.

  First, in the presence of methionine, AdoMet, or AdoHcy, the transcription amount of the cell cycle regulator in the wild type was measured and compared. Specifically, methionine, AdoMet, or AdoHcy was added to a culture medium of wild type, and the transcription amounts of the SWE1 gene and the CLN2 gene were examined by Northern analysis over time. The result is shown in FIG. As shown in FIG. 5 (a), AdoMet and AdoHcy suppressed the transcription of the SWE1 gene and the CLN2 gene.

  Next, methionine, AdoMet, or AdoHcy was added to the culture medium of wild type, and the amounts of SWE1 protein and CLN2 genetic protein were examined by Western analysis over time. The result is shown in FIG. As shown in FIG. 5 (b), AdoMet and AdoHcy decreased the amounts of SWE1 protein and CLN2 protein.

  Next, the DNA content of wild yeast in the presence of methionine, AdoMet, or AdoHcy was measured by FACS analysis. FIG.5 (c) is the result of having measured the DNA content using the sample for 3 hours after adding no addition (-), methionine, AdoMet, or AdoHcy from the left. As a result, accumulation of cells in the G1 phase was observed only when AdoMet was added.

  Next, in order to clarify the effect of AdoMet in the wild yeast body, the DNA content in the presence of methionine, AdoMet, or AdoHcy was calculated using a VPS33-deficient yeast mutant showing abnormalities in vacuoles. Measured by analysis. It is suggested that AdoMet is normally accumulated in the vacuole, thereby eliminating its intracellular effect. As described above, it has been clarified that AdoMet delays the progression of the G1 phase by suppressing transcription of a cell cycle regulator.

Example 6
In order to investigate in more detail at what stage of the cell cycle Sah1-1 is defective, yeast cells are synchronized with the G1 phase using the α-factor described above, and then shifted to a medium without α-factor. Then, the cell cycle progression at 37 ° C. was examined by FACS analysis. Similarly, yeast cells were synchronized with S phase using hydroxyurea (HU), a DNA synthesis inhibitor, and then shifted to a medium containing no HU, and cell cycle progression at 37 ° C. was examined by FACS analysis. The result is shown in FIG.

  As shown in FIG. 6, Sah1-1 synchronized with S phase by HU progressed from S phase to G2 after shifting to 37 ° C. like wild type. On the other hand, sah1-1 synchronized with the G1 phase by an α factor, unlike the wild type, did not progress from the G1 phase to the S phase after shifting to 37 ° C. From this, it became clear that sah1-1 stops at the G1 phase at the limit temperature (37 ° C.) due to a defect at the G1 phase. That is, it was revealed that the point (execution point) where the SAH1 protein actually functions in the cell cycle is the G1 phase.

Example 7
From the results of Examples 1 to 6 described above, it was found that intracellular S-adenosylmethionine delays the cell cycle G1 phase in yeast cells. S-adenosylmethionine was also found to destabilize the cell cycle regulator SWE1. Thus, it was examined whether S-adenosylmethionine can restore the inability to grow in the calcium medium exhibited by the zds1-disrupted strain. As a result, as shown in FIG. 7, S-adenosylmethionine was actually able to restore the inability to grow the zds1-disrupted strain in 300 mM calcium medium.

  So far, S-adenosylmethionine has been reported to cause degradation of MET4 protein involved in methionine metabolism. Therefore, if the above results are caused by degradation of MET4 protein by S-adenosylmethionine, the strain lacking the MET4 gene is caused by S-adenosylmethionine observed in the zds1-disrupted strain. Expected to show no growth recovery. However, by adding S-adenosylmethionine to the medium, the inability to grow on 100 mM (or 300 mM) calcium medium exhibited by the zds1met4 double disruption strain could be recovered. From the above results, S-adenosylmethionine actually recovered the inability to grow on the calcium medium exhibited by the zds1Δ-disrupted strain by suppressing the expression of cell cycle regulators SWE1 and CLN2. Furthermore, it was suggested that this effect is a novel pathway not involving the MET4 protein.

  The yeast mutant according to the present invention can accumulate S-adenosylmethionine in cells. Therefore, according to the yeast mutant of the present invention, S-adenosylmethionine can be produced in large quantities. S-adenosylmethionine is useful as a therapeutic agent for various diseases such as liver disease, depression treatment, osteoarthritis, and cancer treatment. Thus, the present invention is particularly applicable in the pharmaceutical industry.

  It is also of great academic significance because it can be used to elucidate the relationship between intracellular S-adenosylmethionine and cell proliferation (cell cycle).

FIG. 1 is an explanatory diagram showing the outline of the production pathway of S-adenosylmethionine in cells. FIG. 2 is a diagram showing the phenotypes of wild type, zds1Δ, zds1Δsah1-1, and sah1-1, and (a) shows the growth status of various yeasts at 14 ° C., 25 ° C., or 37 ° C. (B) is a photograph showing the cell morphology of various yeasts in the presence of calcium, and (c) is a diagram showing the DNA content of various yeast mutants in the presence of calcium. FIG. 3 is a view showing the expression status of SWE1 gene and CLN2 gene in wild type, zds1Δ, zds1Δsah1-1, and sah1-1, and (a) shows the transcription status of SWE1 gene and CLN2 gene in various yeasts. (B) is a figure which shows the expression condition of SWE1 protein and CLN2 protein in various yeast, (c) is a SWE1 gene on a cell cycle, and CLN2 in wild type and sah1-1. It is a figure which shows the result of having investigated the transcriptional periodicity of the gene. FIG. 4 is a photograph showing the growth of wild type and sah1-1 in the presence of methionine, S-adenosylmethionine, or S-adenosyl-L-homocysteine. FIG. 5 is a diagram showing the expression status of the wild type SWE1 gene and the CLN2 gene in the presence of methionine, S-adenosylmethionine, or S-adenosyl-L-homocysteine, and (a) shows SWE1. (B) is a diagram showing the expression status of the SWE1 protein and the CLN2 protein, and (c) is a diagram showing the expression status of the gene and the CLN2 gene, and (c) is a diagram showing the methionine, S-adenosylmethionine, or S-adenosyl- It is a figure which shows the DNA content of wild type in presence of L-homocysteine, (d) is a figure which shows the DNA content of the VPS33 yeast variant in presence of S-adenosyl methionine. FIG. 6 shows that for wild type and sah1-1, each yeast cell was synchronized with G1 phase or S phase using α-factor or hydroxyurea (HU) and then shifted to 37 ° C. to show cell cycle progression. It is a figure which shows the result investigated by FACS analysis. FIG. 7 is a photograph showing the results of comparison of the growth status of wild type, zds1Δ, and zs1Δmet4Δ at a calcium concentration of 100 mM or 300 mM in the absence of AdoMet and in the presence of AdoMet.

Claims (18)

  A yeast mutant characterized in that it is capable of accumulating S-adenosylmethionine in a cell, wherein a methionine metabolic enzyme is mutated.   The methionine metabolic enzyme is S-adenosylhomocysteine hydrolase, and one or more amino acids are substituted in the amino acid sequence of wild-type S-adenosylhomocysteine hydrolase shown in SEQ ID NO: 3 The yeast mutant according to claim 1, which has a mutant S-adenosylhomocysteine hydrolase comprising an amino acid sequence which is deleted, inserted, and / or added.   The yeast mutant according to claim 2, wherein the amino acid sequence of the mutant S-adenosylhomocysteine hydrolase is the amino acid sequence represented by SEQ ID NO: 4.   The yeast mutant according to claim 2 or 3, which has a gene encoding the mutant S-adenosylhomocysteine hydrolase.   The yeast mutant according to claim 4, wherein the gene encoding the mutant S-adenosylhomocysteine hydrolase comprises the base sequence represented by SEQ ID NO: 2.   The yeast mutant according to any one of claims 1 to 5, wherein the yeast mutant is Sacharomyces cerevisiae FERMP-19715.   A method for producing S-adenosylmethionine, comprising producing S-adenosylmethionine by culturing the yeast mutant according to any one of claims 1 to 6.   A screening method for S-adenosylmethionine high-producing yeast, characterized in that the phenotype shown in the absence of methionine is determined using as an index the recovery in the presence of methionine.   The method for screening a yeast producing high S-adenosylmethionine according to claim 8, wherein the phenotype is temperature sensitive.   A method for screening a yeast producing a high amount of S-adenosylmethionine, which comprises detecting an amino acid sequence of a methionine metabolic enzyme or a mutation in a gene encoding the methionine metabolic enzyme.   The method for screening a yeast producing high S-adenosylmethionine according to claim 10, wherein the methionine metabolic enzyme is S-adenosylhomocysteine hydrolase.   The amino acid sequence of the wild-type S-adenosylhomocysteine hydrolase shown in SEQ ID NO: 3 is composed of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added. Mutant S-adenosylhomocysteine hydrolase.   13. The mutant S-adenosyl homocysteine hydrolase according to claim 12, wherein the amino acid sequence of the mutant S-adenosyl homocysteine hydrolase is the amino acid sequence represented by SEQ ID NO: 4. .   A gene encoding the mutant S-adenosylhomocysteine hydrolase according to claim 12 or 13.   The gene according to claim 14, wherein the gene encoding the mutant S-adenosylhomocysteine hydrolase comprises the base sequence shown in SEQ ID NO: 2.   A recombinant expression vector comprising the gene according to claim 14 or 15.   A method for producing a transformant, wherein the gene according to claim 14 or 15 is introduced into a host cell.   A transformant obtained by the method for producing a transformant according to claim 17.

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