WO2009101945A1 - Method of producing s-adenosylmethionine - Google Patents
Method of producing s-adenosylmethionine Download PDFInfo
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- WO2009101945A1 WO2009101945A1 PCT/JP2009/052240 JP2009052240W WO2009101945A1 WO 2009101945 A1 WO2009101945 A1 WO 2009101945A1 JP 2009052240 W JP2009052240 W JP 2009052240W WO 2009101945 A1 WO2009101945 A1 WO 2009101945A1
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- C—CHEMISTRY; METALLURGY
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- the present invention relates to a method for producing S-adenosylmethionine (hereinafter referred to as SAM).
- SAM is a physiologically active substance that is present throughout the body tissue and functions as a methyl group donor for various methylation reactions in the synthesis and metabolism of hormones, neurotransmitters, phospholipids and proteins. Clinically, it has an effect of enhancing liver function and helping to discharge harmful substances from the body, and has been conventionally known to have therapeutic effects on hepatitis, hyperlipidemia, arteriosclerosis and the like. It is also involved in the production of neurotransmitters, and has recently been used as a treatment and supplement for depression, senile dementia, and arthritis. Thus, as the usefulness of SAM becomes clear, demand has increased in recent years, and a method for manufacturing SAM with higher productivity is desired.
- Patent Document 1 discloses that the function of a phosphatidylcholine biosynthesis enzyme, which is one of the enzymes that consume SAM, is suppressed using molecular biological techniques. Mutant strains have been disclosed, and it has been shown that SAM is highly accumulated inside and outside cells by culturing the mutant strains.
- the objective of this invention is providing the manufacturing method which can mass-produce SAM.
- One enzyme related to SAM is a phosphatidylcholine biosynthesis enzyme. This enzyme catalyzes a series of reactions for synthesizing phosphatidylcholine from phosphatidylethanolamine, specifically, phosphatidylethanolamine methyltransferase that methylates the amino group of phosphatidylethanolamine, and N-methylphosphatidylethanolamine. Refers to N-methylphosphatidylethanolamine methyltransferase that further N-methylates to phosphatidylcholine. In these methylation reactions, SAM is known to be involved as a methyl group donor (FIG. 1).
- the biosynthetic pathway of phosphatidylcholine includes a regenerative pathway (salvage pathway) in addition to the above-mentioned de novo pathway by the phosphatidylcholine biosynthetic enzyme. It is considered that the routes are competing with each other (FIG. 2).
- the present invention has the following gist.
- a method for producing S-adenosylmethionine comprising culturing yeast in a medium containing a choline-based compound and recovering S-adenosylmethionine accumulated in the system.
- the choline-based compound is choline, phosphatidylcholine, phosphocholine, cytidine-5′-diphosphate choline, or acetylcholine.
- the yeast belongs to the genus Saccharomyces.
- the method according to (3) above, wherein the yeast is Saccharomyces cerevisiae.
- the yeast according to (1) wherein the yeast is a mutant having a sequence in which one or more bases are substituted, deleted, inserted and / or added in a gene encoding a phosphatidylcholine synthase.
- Method. (6) The method according to (5) above, wherein the yeast is a cho2 mutant.
- the medium containing the choline compound is a medium containing 10 ⁇ M or more of the choline compound.
- the medium supplemented with the choline compound is a medium containing 0.1 to 100 mM choline compound.
- SAM is accumulated at a high concentration inside and outside the cell, so that it is possible to produce SAM more efficiently and in a larger amount than before. Moreover, SAM productivity can be further improved by using a high-producing strain such as a cho2 mutant as yeast.
- the present invention is a method for improving the productivity of SAM by adding a choline compound to a medium suitable for SAM-producing bacteria.
- the choline-based compound refers to a group of compounds that are decomposed in vivo or easily converted into choline.
- Compounds that can be converted to choline include those in which phosphatidyl group, phosphate group, CDP group, acetic acid group, etc. are ester-bonded to the hydroxyl group of choline, and these compounds are easily decomposed and converted to choline in the living body. Things are known.
- preferred compounds include choline, phosphatidylcholine, phosphocholine, cytidine-5'-diphosphate choline and acetylcholine.
- the addition amount is 500 mM or less, preferably 100 mM or less. Particularly preferred is 1 mM. If it is less than 10 ⁇ M, a sufficient SAM accumulation effect cannot be obtained, and if it exceeds 500 mM, the culture and purification are adversely affected.
- Yeast refers to a unicellular fungus, and examples include spore yeast, basidiomycetous yeast, and incomplete yeast. Of these, spore yeasts such as Saccharomyces, Picha, Hansenula, Zygosaccharomyces are desirable, and Saccharomyces is more desirable. Of these, Saccharomyces cerevisiae is more desirable. Specifically, laboratory yeast, sake yeast, shochu yeast, wine yeast, beer yeast, baker's yeast and the like are included. More preferable yeasts include mutants of yeast having a sequence in which one or more bases are substituted, deleted, inserted and / or added in a gene encoding a phosphatidylcholine synthase. Among them, a typical example is a phosphatidylethanolamine methyltransferase gene (CHO2 gene) mutant (hereinafter also referred to as “cho2 mutant”).
- CHO2 gene phosphatidylethanolamine methyltransferase gene
- SAM production can be produced in high yield by culturing yeast in an appropriate medium, accumulating and accumulating SAM inside and outside the cells, and passing through an appropriate purification step.
- the culture of yeast is not particularly limited, and culture conditions suitable for yeast can be usually used.
- the culture temperature is 20 to 45 ° C. (preferably 25 to 40 ° C.) and the pH during the culture is controlled to 3 to 8 (preferably 5 to 8) for 12 to 150 hours (preferably 16 Up to 120 hours).
- an inorganic or organic acidic or alkaline substance, ammonia gas or the like can be used for pH adjustment.
- a medium suitable for yeast can be used.
- a normal medium containing basic components such as a carbon source and a nitrogen source and inorganic ions and other organic components as required.
- the component selection is not particularly limited as long as yeast is a substance that can be used from past knowledge.
- the carbon source is not limited to monosaccharides such as glucose and fructose, but also disaccharides such as sucrose and lactose, polysaccharides such as cellulose and starch, organic compounds such as ethanol and lactic acid, and crude raw materials such as molasses Etc. are also available.
- inorganic salts such as ammonium salts and nitrates
- nitrogen-containing organic compounds such as amino acids and glucosamine
- organic raw materials such as yeast extract and peptone
- suitable inorganic ion salts, vitamins, minerals, organic compounds, buffer components, antifoaming agents and the like can be added from the knowledge of past culture engineering.
- the amount of methionine added is 0.01 w / v% or more, preferably 0.05 w / v% or more, more preferably 0.1 w / v% or more.
- the addition amount is 0.4 w / v% or less, preferably 0.35 w / v% or less, more preferably 0.3 w / v% or less.
- the addition amount is most preferably in the range of 0.1 to 0.3 w / v%. If the added amount is less than 0.01 w / v%, a sufficient SAM accumulation effect cannot be obtained, and if it exceeds 0.4 w / v%, the growth of the yeast is adversely affected.
- “w / v%” indicates a percentage of mass to volume.
- the extraction and purification of SAM from the cultured medium is not particularly limited.
- recovery of the microbial cell from a culture it can implement by methods, such as centrifugation, precipitation, and filtration.
- yeast can be easily recovered by centrifugation or filtration.
- SAM can be recovered from the obtained cells by physical destruction methods (homogenizer, glass bead crushing, freeze-thawing, etc.) or chemical destruction methods (solvent treatment, acid, base treatment, osmotic pressure treatment, enzyme treatment, etc.) ).
- the purified SAM can be purified by existing purification methods (solvent extraction, column chromatography, salt precipitation, etc.).
- SAM can be purified by using acidic ion exchange chromatography, and can be recovered as a solid by salt precipitation by adding cold acetone.
- Example Culture was carried out using laboratory yeast (Saccharomyces cerevisiae BY20592), its cho2 mutant, and sake yeast (Association No. 7 yeast) as parent strains, and the SAM content accumulated in the cells was measured. All laboratory yeast strains used in this test were obtained from the Yeast Genetic Resource Center.
- a vector for target gene destruction was produced using pAUR135 vector manufactured by Takara Bio Inc. Specifically, the EcoR I-Sma I restriction enzyme digestion product of the pAUR135 vector and a partial PCR amplification product of CHO2 (primary A and B (Table 1, SEQ ID NO: 1) using the genomic DNA of laboratory yeast BY2041 as a template) The EcoR I restriction enzyme digestion product of the sequence amplified by the PCR reaction using 1 and 2) was mixed and ligated with T4 DNA ligase to prepare a CHO2 disruption vector.
- This PCR reaction product was directly transformed into Escherichia coli DH5 ⁇ to obtain Escherichia coli carrying the CHO2 disruption vector into which the target point mutation was introduced. Plasmid purification was performed from this E. coli culture according to a conventional method to prepare a CHO2 disruption vector into which a necessary amount of point mutation was introduced (hereinafter referred to as “CHO2 mutation introduction vector”).
- the mutation was introduced into the CHO2 gene of the yeast genome using the above CHO2 mutagenesis vector.
- laboratory yeast Sacharomyces cerevisiae BY20592
- the above vector was introduced by the lithium acetate method for transformation.
- This transformed yeast is cultured overnight in a YPD liquid medium, then applied to a 0.5 ⁇ g / ml aureobasidin A-containing YPD solid medium, and the introduced vector is inserted into the CHO2 gene site for the grown colonies.
- YPD liquid medium a 0.5 ⁇ g / ml aureobasidin A-containing YPD solid medium
- the colonies were spread on a YP-Galactose solid medium (2 w / v% peptone, 1 w / v% yeast extract, 2 w / v% galactose, 2 w / v% purified agar) and cultured at 28 ° C. for 3 days.
- the pAUR135 vector has galactose-induced lethality
- the growing colony is a revertant in which the vector sequence has been dropped, and the stop codon introduced into the cho2 mutagenesis vector is introduced into the genome.
- 5 ml of SAM fermentation medium (5 w / v% glucose, 1 w / v% peptone, 0.5 w / v% yeast extract, 0.4 w / v% KH 2 PO 4 , 0.2 w / v% K in a 50 ml centrifuge tube 2 HPO 4 , 0.05 w / v% Mg 2 SO 4 .H 2 O, 0.15 w / v% L-methionine, pH 6.0) is dispensed, and the final concentration is 10 ⁇ M to 100 mM. Choline was added at every double concentration. Each of these media was inoculated with a yeast strain and immersed in culture at 28 ° C. for 72 hours to obtain a sufficient amount of culture.
- Quantification was performed by comparison with a standard product using UV absorption of 260 nm as an index using HPLC.
- Analytical conditions are as follows: Equipment used: Waters 2690 Separation Module Waters 2487 Dual Absorbance Detector system, Column used: cosmosil packed column 5C18-MS (4.6id x 250mm), Elution solvent: 5v / v% Methanol-95v / v% 0.2M KH 2 PO 4 solution, flow rate: 1 ml / min, column temperature: 25 ° C.
- the present invention contributes to mass supply of SAM widely used in the medical field and others including therapeutic drugs and supplements. It should be noted that the entire contents of the specification, claims, drawings and abstract of Japanese Patent Application No. 2008-030672, filed on February 12, 2008, are cited herein as disclosure of the specification of the present invention. Incorporated.
Abstract
Disclosed is a production method whereby SAM can be mass produced. A method of producing S-adenosylmethionine which comprises culturing a yeast in a medium containing a choline compound and collecting the S-adenosylmethionine thus accumulated in the system.
Description
本発明は、Ｓ－アデノシルメチオニン（Ｓ－adenosylmethionine：以下、ＳＡＭという）の製造方法に関する。
The present invention relates to a method for producing S-adenosylmethionine (hereinafter referred to as SAM).
ＳＡＭは、生体組織全体に存在し、ホルモン、神経伝達物質、リン脂質及びタンパク質の合成および代謝における様々なメチル化反応のメチル基供与体として機能する生理活性物質である。臨床的には、肝臓機能を高め、有害物質を体内から排出するのを助ける作用があり、従来から肝炎、高脂血症、動脈硬化症などに対する治療効果があることが知られている。また、神経伝達物質の生成にも関与し、近年ではうつ病、老人性痴呆症、関節炎の治療薬やサプリメントとしても使われている。このようにＳＡＭの有用性が明らかになるに従って近年需要が拡大し、さらに生産性の高いＳＡＭの製造方法が希求されている。
SAM is a physiologically active substance that is present throughout the body tissue and functions as a methyl group donor for various methylation reactions in the synthesis and metabolism of hormones, neurotransmitters, phospholipids and proteins. Clinically, it has an effect of enhancing liver function and helping to discharge harmful substances from the body, and has been conventionally known to have therapeutic effects on hepatitis, hyperlipidemia, arteriosclerosis and the like. It is also involved in the production of neurotransmitters, and has recently been used as a treatment and supplement for depression, senile dementia, and arthritis. Thus, as the usefulness of SAM becomes clear, demand has increased in recent years, and a method for manufacturing SAM with higher productivity is desired.
現在、ＳＡＭの工業的生産は微生物培養によって行われており、主に酵母が使用されている。生産に使用される酵母は、過去の経験や研究からＳＡＭを高蓄積することが知られている既存の菌株が使用されており、ビタミン類、アミノ酸、各種塩などの基本的な栄養成分や培養条件を検討することによりその生産性の向上が図られてきた。しかし、効率的にＳＡＭ蓄積株を選抜する方法がなく、又、培養条件で飛躍的にＳＡＭの生産性を向上させることには限界があった。
Currently, industrial production of SAM is performed by microbial culture, and yeast is mainly used. For the yeast used for production, existing strains that are known to accumulate high SAM from past experience and research are used, and basic nutritional components such as vitamins, amino acids, various salts, and culture The productivity has been improved by examining the conditions. However, there is no method for efficiently selecting SAM-accumulating strains, and there has been a limit to dramatically improving SAM productivity under culture conditions.
一方、近年のバイオテクノロジーの進歩により、微生物の代謝経路や遺伝子の情報などが整備され、ＳＡＭの代謝、蓄積に関与する遺伝子を推定し、直接特定遺伝子を加工する事が可能となり、新たなアプローチで変異株を作製したり、製造方法を改良することが可能となった。
上記アプローチによりＳＡＭの高蓄積変異株の作製を行った例として、特許文献１にはＳＡＭを消費する酵素の一つであるホスファチジルコリン生合成系酵素の機能を分子生物学的手法を用いて抑制した変異株が開示されており、該変異株を培養することにより細胞内外にＳＡＭが高蓄積することが示されている。
国際公開ＷＯ２００８／０２０５９５号公報
On the other hand, recent advances in biotechnology have improved information on microbial metabolic pathways and genes, making it possible to estimate genes involved in SAM metabolism and accumulation and directly process specific genes. It became possible to produce mutants and improve the production method.
As an example of producing a SAM high-accumulation mutant strain by the above approach,Patent Document 1 discloses that the function of a phosphatidylcholine biosynthesis enzyme, which is one of the enzymes that consume SAM, is suppressed using molecular biological techniques. Mutant strains have been disclosed, and it has been shown that SAM is highly accumulated inside and outside cells by culturing the mutant strains.
International Publication WO2008 / 020595
上記アプローチによりＳＡＭの高蓄積変異株の作製を行った例として、特許文献１にはＳＡＭを消費する酵素の一つであるホスファチジルコリン生合成系酵素の機能を分子生物学的手法を用いて抑制した変異株が開示されており、該変異株を培養することにより細胞内外にＳＡＭが高蓄積することが示されている。
As an example of producing a SAM high-accumulation mutant strain by the above approach,
本発明の目的は、ＳＡＭを大量生産し得る製造方法を提供することにある。
The objective of this invention is providing the manufacturing method which can mass-produce SAM.
ＳＡＭに関連する酵素の一つとしてホスファチジルコリン生合成系酵素がある。本酵素は、ホスファチジルエタノールアミンからホスファチジルコリンを合成する一連の反応を触媒する酵素で、具体的にはホスファチジルエタノールアミンのアミノ基をメチル化するホスファチジルエタノールアミンメチル基転移酵素、およびＮ-メチルホスファチジルエタノールアミンをホスファチジルコリンとなるように更にＮ-メチル化するN-メチルホスファチジルエタノールアミンメチル基転移酵素を指す。これらメチル化反応においてＳＡＭはメチル基供与体として関与する事が知られている（図１）。
上記の生合成経路において、ホスファチジルコリン生合成系酵素をコードする遺伝子を変異させてその酵素機能を消失させることにより、ホスファチジルエタノールアミンからホスファチジルコリンの生合成経路におけるＳＡＭの消費が抑えられ、結果として菌体内外にＳＡＭが蓄積することが判明した（特許文献１）。
一方、ホスファチジルコリンを生合成する経路は上記ホスファチジルコリン生合成系酵素による新規生合成系経路（de novo pathway）以外に、再生系経路(salvage pathway)が存在し、ホスファチジルコリンの生合成ではこの２つの生合成経路が互いに競合していると考えられる（図２）。即ち、一方の生合成系を活性化すれば他方は抑制される。
そこで、再生系経路を活性化させることで新規生合成系経路を抑制し、結果としてＳＡＭの消費を抑えることができるのではないかとの仮説をたて、培地中に再生系の基質たるコリン系化合物を添加してＳＡＭ生産株を培養した。その結果、細胞内外にＳＡＭが高蓄積する事を見出し、本発明を完成した。 One enzyme related to SAM is a phosphatidylcholine biosynthesis enzyme. This enzyme catalyzes a series of reactions for synthesizing phosphatidylcholine from phosphatidylethanolamine, specifically, phosphatidylethanolamine methyltransferase that methylates the amino group of phosphatidylethanolamine, and N-methylphosphatidylethanolamine. Refers to N-methylphosphatidylethanolamine methyltransferase that further N-methylates to phosphatidylcholine. In these methylation reactions, SAM is known to be involved as a methyl group donor (FIG. 1).
In the above biosynthetic pathway, by mutating a gene encoding a phosphatidylcholine biosynthetic enzyme and losing its enzyme function, consumption of SAM in the biosynthetic pathway of phosphatidylcholine from phosphatidylethanolamine can be suppressed, resulting in bacterial cells It has been found that SAM accumulates inside and outside (Patent Document 1).
On the other hand, the biosynthetic pathway of phosphatidylcholine includes a regenerative pathway (salvage pathway) in addition to the above-mentioned de novo pathway by the phosphatidylcholine biosynthetic enzyme. It is considered that the routes are competing with each other (FIG. 2). That is, if one biosynthetic system is activated, the other is suppressed.
Therefore, by activating the regenerative pathway, the hypothesis that the biosynthetic pathway can be suppressed and, as a result, the consumption of SAM can be suppressed, the choline system that is the substrate of the regenerative system in the medium. The SAM production strain was cultured by adding the compound. As a result, it was found that SAM was highly accumulated inside and outside the cell, and the present invention was completed.
上記の生合成経路において、ホスファチジルコリン生合成系酵素をコードする遺伝子を変異させてその酵素機能を消失させることにより、ホスファチジルエタノールアミンからホスファチジルコリンの生合成経路におけるＳＡＭの消費が抑えられ、結果として菌体内外にＳＡＭが蓄積することが判明した（特許文献１）。
一方、ホスファチジルコリンを生合成する経路は上記ホスファチジルコリン生合成系酵素による新規生合成系経路（de novo pathway）以外に、再生系経路(salvage pathway)が存在し、ホスファチジルコリンの生合成ではこの２つの生合成経路が互いに競合していると考えられる（図２）。即ち、一方の生合成系を活性化すれば他方は抑制される。
そこで、再生系経路を活性化させることで新規生合成系経路を抑制し、結果としてＳＡＭの消費を抑えることができるのではないかとの仮説をたて、培地中に再生系の基質たるコリン系化合物を添加してＳＡＭ生産株を培養した。その結果、細胞内外にＳＡＭが高蓄積する事を見出し、本発明を完成した。 One enzyme related to SAM is a phosphatidylcholine biosynthesis enzyme. This enzyme catalyzes a series of reactions for synthesizing phosphatidylcholine from phosphatidylethanolamine, specifically, phosphatidylethanolamine methyltransferase that methylates the amino group of phosphatidylethanolamine, and N-methylphosphatidylethanolamine. Refers to N-methylphosphatidylethanolamine methyltransferase that further N-methylates to phosphatidylcholine. In these methylation reactions, SAM is known to be involved as a methyl group donor (FIG. 1).
In the above biosynthetic pathway, by mutating a gene encoding a phosphatidylcholine biosynthetic enzyme and losing its enzyme function, consumption of SAM in the biosynthetic pathway of phosphatidylcholine from phosphatidylethanolamine can be suppressed, resulting in bacterial cells It has been found that SAM accumulates inside and outside (Patent Document 1).
On the other hand, the biosynthetic pathway of phosphatidylcholine includes a regenerative pathway (salvage pathway) in addition to the above-mentioned de novo pathway by the phosphatidylcholine biosynthetic enzyme. It is considered that the routes are competing with each other (FIG. 2). That is, if one biosynthetic system is activated, the other is suppressed.
Therefore, by activating the regenerative pathway, the hypothesis that the biosynthetic pathway can be suppressed and, as a result, the consumption of SAM can be suppressed, the choline system that is the substrate of the regenerative system in the medium. The SAM production strain was cultured by adding the compound. As a result, it was found that SAM was highly accumulated inside and outside the cell, and the present invention was completed.
すなわち、本発明は、以下の要旨を有する。
（１）コリン系化合物を添加した培地で酵母を培養し、系内に蓄積したＳ－アデノシルメチオニンを回収することを特徴とするＳ－アデノシルメチオニンを生産する方法。
（２）前記コリン系化合物がコリン、ホスファチジルコリン、ホスホコリン、シチジン－５’－ジリン酸コリン、又はアセチルコリンである上記（１）に記載の方法。
（３）前記酵母がサッカロミセス（Saccharomyces）属である上記（１）に記載の方法。
（４）前記酵母がサッカロミセス セレヴィシエ（Saccharomyces cerevisiae）である上記（３）に記載の方法。
（５）前記酵母が、ホスファチジルコリン合成系酵素をコードする遺伝子において、１個またはそれ以上の塩基が置換、欠失、挿入および／または付加した配列を有する変異体である上記（１）に記載の方法。
（６）前記酵母がcho2変異体である上記（５）に記載の方法。
（７）前記コリン系化合物を添加した培地が、１０μＭ以上のコリン系化合物を含む培地である上記（１）に記載の方法。
（８）前記コリン系化合物を添加した培地が、０．１～１００ｍＭのコリン系化合物を含む培地である上記（７）に記載の方法に関する。
（９）前記コリン系化合物を添加した培地が、メチオニンを含む培地である上記（１）に記載の方法。
（１０）前記コリン系化合物を添加した培地が、０．０１w/v％以上のメチオニンを含む培地である上記（９）に記載の方法。 That is, the present invention has the following gist.
(1) A method for producing S-adenosylmethionine, comprising culturing yeast in a medium containing a choline-based compound and recovering S-adenosylmethionine accumulated in the system.
(2) The method according to (1) above, wherein the choline-based compound is choline, phosphatidylcholine, phosphocholine, cytidine-5′-diphosphate choline, or acetylcholine.
(3) The method according to (1) above, wherein the yeast belongs to the genus Saccharomyces.
(4) The method according to (3) above, wherein the yeast is Saccharomyces cerevisiae.
(5) The yeast according to (1), wherein the yeast is a mutant having a sequence in which one or more bases are substituted, deleted, inserted and / or added in a gene encoding a phosphatidylcholine synthase. Method.
(6) The method according to (5) above, wherein the yeast is a cho2 mutant.
(7) The method according to (1) above, wherein the medium containing the choline compound is a medium containing 10 μM or more of the choline compound.
(8) The method according to (7) above, wherein the medium supplemented with the choline compound is a medium containing 0.1 to 100 mM choline compound.
(9) The method according to (1) above, wherein the medium supplemented with the choline compound is a medium containing methionine.
(10) The method according to (9) above, wherein the medium supplemented with the choline compound is a medium containing 0.01 w / v% or more methionine.
（１）コリン系化合物を添加した培地で酵母を培養し、系内に蓄積したＳ－アデノシルメチオニンを回収することを特徴とするＳ－アデノシルメチオニンを生産する方法。
（２）前記コリン系化合物がコリン、ホスファチジルコリン、ホスホコリン、シチジン－５’－ジリン酸コリン、又はアセチルコリンである上記（１）に記載の方法。
（３）前記酵母がサッカロミセス（Saccharomyces）属である上記（１）に記載の方法。
（４）前記酵母がサッカロミセス セレヴィシエ（Saccharomyces cerevisiae）である上記（３）に記載の方法。
（５）前記酵母が、ホスファチジルコリン合成系酵素をコードする遺伝子において、１個またはそれ以上の塩基が置換、欠失、挿入および／または付加した配列を有する変異体である上記（１）に記載の方法。
（６）前記酵母がcho2変異体である上記（５）に記載の方法。
（７）前記コリン系化合物を添加した培地が、１０μＭ以上のコリン系化合物を含む培地である上記（１）に記載の方法。
（８）前記コリン系化合物を添加した培地が、０．１～１００ｍＭのコリン系化合物を含む培地である上記（７）に記載の方法に関する。
（９）前記コリン系化合物を添加した培地が、メチオニンを含む培地である上記（１）に記載の方法。
（１０）前記コリン系化合物を添加した培地が、０．０１w/v％以上のメチオニンを含む培地である上記（９）に記載の方法。 That is, the present invention has the following gist.
(1) A method for producing S-adenosylmethionine, comprising culturing yeast in a medium containing a choline-based compound and recovering S-adenosylmethionine accumulated in the system.
(2) The method according to (1) above, wherein the choline-based compound is choline, phosphatidylcholine, phosphocholine, cytidine-5′-diphosphate choline, or acetylcholine.
(3) The method according to (1) above, wherein the yeast belongs to the genus Saccharomyces.
(4) The method according to (3) above, wherein the yeast is Saccharomyces cerevisiae.
(5) The yeast according to (1), wherein the yeast is a mutant having a sequence in which one or more bases are substituted, deleted, inserted and / or added in a gene encoding a phosphatidylcholine synthase. Method.
(6) The method according to (5) above, wherein the yeast is a cho2 mutant.
(7) The method according to (1) above, wherein the medium containing the choline compound is a medium containing 10 μM or more of the choline compound.
(8) The method according to (7) above, wherein the medium supplemented with the choline compound is a medium containing 0.1 to 100 mM choline compound.
(9) The method according to (1) above, wherein the medium supplemented with the choline compound is a medium containing methionine.
(10) The method according to (9) above, wherein the medium supplemented with the choline compound is a medium containing 0.01 w / v% or more methionine.
本発明の生産方法では、細胞内外にＳＡＭを高濃度で蓄積するので、従来よりも効率的かつ大量にＳＡＭを生産する事が可能となる。また、酵母としてcho2変異体等の高生産株を用いることにより、更にＳＡＭの生産性を向上させることができる。
In the production method of the present invention, SAM is accumulated at a high concentration inside and outside the cell, so that it is possible to produce SAM more efficiently and in a larger amount than before. Moreover, SAM productivity can be further improved by using a high-producing strain such as a cho2 mutant as yeast.
本発明の一形態について説明するが、本発明はこれに限定されるものではない。
本発明はＳＡＭ生産菌に好適な培地に対して、コリン系化合物を添加することによりＳＡＭの生産性を向上させる方法である。 Although one form of this invention is demonstrated, this invention is not limited to this.
The present invention is a method for improving the productivity of SAM by adding a choline compound to a medium suitable for SAM-producing bacteria.
本発明はＳＡＭ生産菌に好適な培地に対して、コリン系化合物を添加することによりＳＡＭの生産性を向上させる方法である。 Although one form of this invention is demonstrated, this invention is not limited to this.
The present invention is a method for improving the productivity of SAM by adding a choline compound to a medium suitable for SAM-producing bacteria.
コリン系化合物とは、コリン又は生体内で分解し容易にコリンに転換される化合物の一群をいう。コリンに転換される化合物として、コリンの水酸基にホスファチジル基、リン酸基、CDP基、酢酸基などがエステル結合したものなどが挙げられ、これら化合物は生物体内で容易に分解しコリンへ変換される事が知られている。コリン系化合物の内、好ましい化合物としては、コリン、ホスファチジルコリン、ホスホコリン、シチジン－５’－ジリン酸コリンやアセチルコリンが挙げられる。コリン系化合物の添加量は、１０μＭ（＝０．０１ｍＭ）以上であり、好ましくは０．１ｍＭ以上である。該添加量は５００ｍＭ以下であり、好ましくは１００ｍＭ以下である。特に好ましくは１ｍＭである。１０μＭ未満であれば、十分なＳＡＭの蓄積効果が得られず、また、５００ｍＭ超であると培養や精製に悪影響を与えるため好ましくない。
The choline-based compound refers to a group of compounds that are decomposed in vivo or easily converted into choline. Compounds that can be converted to choline include those in which phosphatidyl group, phosphate group, CDP group, acetic acid group, etc. are ester-bonded to the hydroxyl group of choline, and these compounds are easily decomposed and converted to choline in the living body. Things are known. Among the choline compounds, preferred compounds include choline, phosphatidylcholine, phosphocholine, cytidine-5'-diphosphate choline and acetylcholine. The addition amount of the choline compound is 10 μM (= 0.01 mM) or more, preferably 0.1 mM or more. The addition amount is 500 mM or less, preferably 100 mM or less. Particularly preferred is 1 mM. If it is less than 10 μM, a sufficient SAM accumulation effect cannot be obtained, and if it exceeds 500 mM, the culture and purification are adversely affected.
酵母とは、単細胞の真菌を示し、有胞子酵母、担子菌酵母、不完全酵母などが挙げられる。この中でもＳａｃｃｈａｒｏｍｙｃｅｓ属、Ｐｉｃｈａ属、Ｈａｎｓｅｎｕｌａ属、Ｚｙｇｏｓａｃｃｈａｒｏｍｙｃｅｓ属など有胞子酵母が望ましく、より望ましくはＳａｃｃｈａｒｏｍｙｃｅｓ属である。中でもＳａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅが更に望ましい。具体的には、実験室酵母、清酒酵母、焼酎酵母、ワイン酵母、ビール酵母、パン酵母などが含まれる。又、より好ましい酵母としては、ホスファチジルコリン合成系酵素をコードする遺伝子において、１個またはそれ以上の塩基が置換、欠失、挿入および／または付加した配列を有する酵母の変異体が挙げられる。なかでも、ホスファチジルエタノールアミンメチル基転移酵素遺伝子（CHO2遺伝子）の変異体（以下、「cho2変異体」ともいう）が代表的な例として挙げられる。
Yeast refers to a unicellular fungus, and examples include spore yeast, basidiomycetous yeast, and incomplete yeast. Of these, spore yeasts such as Saccharomyces, Picha, Hansenula, Zygosaccharomyces are desirable, and Saccharomyces is more desirable. Of these, Saccharomyces cerevisiae is more desirable. Specifically, laboratory yeast, sake yeast, shochu yeast, wine yeast, beer yeast, baker's yeast and the like are included. More preferable yeasts include mutants of yeast having a sequence in which one or more bases are substituted, deleted, inserted and / or added in a gene encoding a phosphatidylcholine synthase. Among them, a typical example is a phosphatidylethanolamine methyltransferase gene (CHO2 gene) mutant (hereinafter also referred to as “cho2 mutant”).
ＳＡＭ生産は、酵母を適切な培地で培養し、ＳＡＭを菌体内外に生産蓄積せしめ、適切な精製工程を経ることによって、高収率で生産することができる。
SAM production can be produced in high yield by culturing yeast in an appropriate medium, accumulating and accumulating SAM inside and outside the cells, and passing through an appropriate purification step.
酵母の培養については特に限定されることはなく、培養条件としては通常、酵母に対して好適な条件を用いることができる。例えば、培養温度を２０～４５℃（好ましくは２５～４０℃）、培養中のｐＨを３～８（好ましくは５～８）に制御した好気的条件下で１２～１５０時間（好ましくは１６～１２０時間）培養することができる。尚、ｐＨ調整には無機あるいは有機の酸性あるいはアルカリ性物質、更にアンモニアガス等を使用することができる。
The culture of yeast is not particularly limited, and culture conditions suitable for yeast can be usually used. For example, the culture temperature is 20 to 45 ° C. (preferably 25 to 40 ° C.) and the pH during the culture is controlled to 3 to 8 (preferably 5 to 8) for 12 to 150 hours (preferably 16 Up to 120 hours). In addition, an inorganic or organic acidic or alkaline substance, ammonia gas or the like can be used for pH adjustment.
培地についても同様に、酵母に対して好適な培地を用いる事ができる。例えば、炭素源、窒素源などの基本成分と、無機イオン及び必要に応じその他の有機成分とを含有する通常の培地である。その成分選択は過去の知見から酵母が利用可能な物質とされていれば、特に限定されるものではない。例えば、炭素源としてはグルコースやフルクトースの様な単糖に限らず、ショ糖や乳糖などの二糖、セルロースやスターチなどの多糖、エタノールや乳酸などの有機化合物、さらには廃糖蜜など粗精製原料なども利用可能である。窒素源に関しても、アンモニウム塩や硝酸塩などの無機塩類だけでなく、アミノ酸やグルコサミンなどの含窒素有機化合物、酵母抽出物やペプトンなどの有機原料なども利用可能である。これらの基本成分に加え、過去の培養工学の知見から好適な無機イオン塩、ビタミン、ミネラル、有機化合物、緩衝成分、消泡剤などを添加することができる。さらにSAM生産にとって望ましくは、メチオニン添加培地を使用することによって、更なるＳＡＭの大量取得が可能となる。
Similarly, a medium suitable for yeast can be used. For example, a normal medium containing basic components such as a carbon source and a nitrogen source and inorganic ions and other organic components as required. The component selection is not particularly limited as long as yeast is a substance that can be used from past knowledge. For example, the carbon source is not limited to monosaccharides such as glucose and fructose, but also disaccharides such as sucrose and lactose, polysaccharides such as cellulose and starch, organic compounds such as ethanol and lactic acid, and crude raw materials such as molasses Etc. are also available. Regarding the nitrogen source, not only inorganic salts such as ammonium salts and nitrates but also nitrogen-containing organic compounds such as amino acids and glucosamine, organic raw materials such as yeast extract and peptone can be used. In addition to these basic components, suitable inorganic ion salts, vitamins, minerals, organic compounds, buffer components, antifoaming agents and the like can be added from the knowledge of past culture engineering. Furthermore, it is desirable for SAM production to use a methionine-added medium, which enables further mass acquisition of SAM.
メチオニンの添加量としては０．０１w/v％以上であり、好ましくは０．０５w/v％以上、更に好ましくは０．１w/v％以上である。該添加量は０．４w/v％以下、好ましくは０．３５w/v％以下、更に好ましくは０．３w/v％以下である。該添加量は０．１～０．３w/v％の範囲内が最も好ましい。該添加量が０．０１w/v％未満では、十分なSAMの蓄積効果が得られず、また、０．４w/v％超では、酵母の生育に悪影響を与えるため好ましくない。
なお、ここで、「w/v％」とは、質量対容量の百分率を示す。 The amount of methionine added is 0.01 w / v% or more, preferably 0.05 w / v% or more, more preferably 0.1 w / v% or more. The addition amount is 0.4 w / v% or less, preferably 0.35 w / v% or less, more preferably 0.3 w / v% or less. The addition amount is most preferably in the range of 0.1 to 0.3 w / v%. If the added amount is less than 0.01 w / v%, a sufficient SAM accumulation effect cannot be obtained, and if it exceeds 0.4 w / v%, the growth of the yeast is adversely affected.
Here, “w / v%” indicates a percentage of mass to volume.
なお、ここで、「w/v％」とは、質量対容量の百分率を示す。 The amount of methionine added is 0.01 w / v% or more, preferably 0.05 w / v% or more, more preferably 0.1 w / v% or more. The addition amount is 0.4 w / v% or less, preferably 0.35 w / v% or less, more preferably 0.3 w / v% or less. The addition amount is most preferably in the range of 0.1 to 0.3 w / v%. If the added amount is less than 0.01 w / v%, a sufficient SAM accumulation effect cannot be obtained, and if it exceeds 0.4 w / v%, the growth of the yeast is adversely affected.
Here, “w / v%” indicates a percentage of mass to volume.
培養後の培地からのＳＡＭの抽出及び精製については、特に限定されることは無い。例えば、培養物からの菌体の回収については、遠心、沈殿、ろ過といった方法で実施することができる。例えば酵母では遠心やろ過により容易に回収が可能である。又、得られた菌体からのＳＡＭの回収は、物理的破壊法（ホモジナイザー、ガラスビーズ破砕、凍結融解など）や化学的破壊法（溶剤処理、酸、塩基処理、浸透圧処理、酵素処理など）によって行なうことができる。例えば酵母では酸処理により容易にSAMは溶出する。さらに、抽出したＳＡＭの精製については、既存の精製方法（溶媒抽出、カラムクロマトグラフィー、塩沈降など）によって実施することができる。具体的にはSAMは酸性のイオン交換クロマトグラフィーを用いる事で精製は可能であり、冷アセトンを添加することで塩沈降によって固体として回収することが可能である。これらの方法は、必要に応じて適宜組み合わせて実施することが可能である。
The extraction and purification of SAM from the cultured medium is not particularly limited. For example, about the collection | recovery of the microbial cell from a culture, it can implement by methods, such as centrifugation, precipitation, and filtration. For example, yeast can be easily recovered by centrifugation or filtration. In addition, SAM can be recovered from the obtained cells by physical destruction methods (homogenizer, glass bead crushing, freeze-thawing, etc.) or chemical destruction methods (solvent treatment, acid, base treatment, osmotic pressure treatment, enzyme treatment, etc.) ). For example, in yeast, SAM is easily eluted by acid treatment. Furthermore, the purified SAM can be purified by existing purification methods (solvent extraction, column chromatography, salt precipitation, etc.). Specifically, SAM can be purified by using acidic ion exchange chromatography, and can be recovered as a solid by salt precipitation by adding cold acetone. These methods can be implemented in appropriate combination as required.
以下、本発明の具体的な実施例について述べるが、本発明はこれに限定されるものではない。
Hereinafter, specific examples of the present invention will be described, but the present invention is not limited thereto.
実施例
実験室酵母（Saccharomyces cerevisiae BY20592）およびそのcho2変異体、清酒酵母（協会7号酵母）をそれぞれ親株として培養を行い、菌体内に蓄積されたSAM含量を測定した。なお、本試験で用いた実験室酵母株は全て酵母遺伝資源センターより入手した。 Example Culture was carried out using laboratory yeast (Saccharomyces cerevisiae BY20592), its cho2 mutant, and sake yeast (Association No. 7 yeast) as parent strains, and the SAM content accumulated in the cells was measured. All laboratory yeast strains used in this test were obtained from the Yeast Genetic Resource Center.
実験室酵母（Saccharomyces cerevisiae BY20592）およびそのcho2変異体、清酒酵母（協会7号酵母）をそれぞれ親株として培養を行い、菌体内に蓄積されたSAM含量を測定した。なお、本試験で用いた実験室酵母株は全て酵母遺伝資源センターより入手した。 Example Culture was carried out using laboratory yeast (Saccharomyces cerevisiae BY20592), its cho2 mutant, and sake yeast (Association No. 7 yeast) as parent strains, and the SAM content accumulated in the cells was measured. All laboratory yeast strains used in this test were obtained from the Yeast Genetic Resource Center.
（１）cho2変異体の作製
標的遺伝子破壊用のベクターはタカラバイオ社製 pAUR135ベクターを利用して作製した。具体的には、pAUR135ベクターのEcoR I－Sma I制限酵素切断処理物と、CHO2の一部のPCR増幅物（実験室酵母BY2041のゲノムDNAを鋳型に、プライマーＡ、Ｂ（表１、配列番号１及び２）を用いたPCR反応によって増幅された配列）のEcoR I制限酵素切断処理物を混合し、T4DNAリガーゼでライゲーションして、CHO2破壊用ベクターを作製した。さらに、このベクターをクローニングするために大腸菌DH5αへ導入し、この大腸菌培養物から常法に従いプラスミド精製を行うことで、必要量の遺伝子破壊用ベクターを調製した。 (1) Production of cho2 mutant A vector for target gene destruction was produced using pAUR135 vector manufactured by Takara Bio Inc. Specifically, the EcoR I-Sma I restriction enzyme digestion product of the pAUR135 vector and a partial PCR amplification product of CHO2 (primary A and B (Table 1, SEQ ID NO: 1) using the genomic DNA of laboratory yeast BY2041 as a template) The EcoR I restriction enzyme digestion product of the sequence amplified by the PCR reaction using 1 and 2) was mixed and ligated with T4 DNA ligase to prepare a CHO2 disruption vector. Furthermore, in order to clone this vector, it introduce | transduced into colon_bacillus | E._coli DH5 (alpha), By carrying out the plasmid purification from this colon_bacillus | E._coli culture | cultivation according to a conventional method, the vector of the gene destruction required quantity was prepared.
標的遺伝子破壊用のベクターはタカラバイオ社製 pAUR135ベクターを利用して作製した。具体的には、pAUR135ベクターのEcoR I－Sma I制限酵素切断処理物と、CHO2の一部のPCR増幅物（実験室酵母BY2041のゲノムDNAを鋳型に、プライマーＡ、Ｂ（表１、配列番号１及び２）を用いたPCR反応によって増幅された配列）のEcoR I制限酵素切断処理物を混合し、T4DNAリガーゼでライゲーションして、CHO2破壊用ベクターを作製した。さらに、このベクターをクローニングするために大腸菌DH5αへ導入し、この大腸菌培養物から常法に従いプラスミド精製を行うことで、必要量の遺伝子破壊用ベクターを調製した。 (1) Production of cho2 mutant A vector for target gene destruction was produced using pAUR135 vector manufactured by Takara Bio Inc. Specifically, the EcoR I-Sma I restriction enzyme digestion product of the pAUR135 vector and a partial PCR amplification product of CHO2 (primary A and B (Table 1, SEQ ID NO: 1) using the genomic DNA of laboratory yeast BY2041 as a template) The EcoR I restriction enzyme digestion product of the sequence amplified by the PCR reaction using 1 and 2) was mixed and ligated with T4 DNA ligase to prepare a CHO2 disruption vector. Furthermore, in order to clone this vector, it introduce | transduced into colon_bacillus | E._coli DH5 (alpha), By carrying out the plasmid purification from this colon_bacillus | E._coli culture | cultivation according to a conventional method, the vector of the gene destruction required quantity was prepared.
前項で作製したCHO2破壊用ベクターに挿入されているCHO2遺伝子の部分配列に対して、CDSに相当する配列でストップコドンが発生するように点突然変異を設計したプライマーC、D（表１、配列番号３及び４）を用いてCHO2破壊用ベクターの全長を増幅した。このPCR反応物をそのまま大腸菌DH5αへ形質転換し、目的の点突然変異が導入されたCHO2破壊用ベクターを保持した大腸菌を得た。この大腸菌培養物から常法に従いプラスミド精製を行い、必要量の点突然変異が導入されたCHO2破壊用ベクター（以下、「CHO2変異導入ベクター」という）を作製した。
Primers C and D designed for point mutation so that a stop codon is generated in the sequence corresponding to CDS for the partial sequence of the CHO2 gene inserted in the CHO2 disruption vector prepared in the previous section (Table 1, sequence) Numbers 3 and 4) were used to amplify the full length of the CHO2 disruption vector. This PCR reaction product was directly transformed into Escherichia coli DH5α to obtain Escherichia coli carrying the CHO2 disruption vector into which the target point mutation was introduced. Plasmid purification was performed from this E. coli culture according to a conventional method to prepare a CHO2 disruption vector into which a necessary amount of point mutation was introduced (hereinafter referred to as “CHO2 mutation introduction vector”).
上記のCHO2変異導入ベクターを用いて酵母ゲノムのCHO2遺伝子への変異導入を行った。具体的には、親株として実験室酵母（Saccharomyces cerevisiae BY20592）を用い、酢酸リチウム法によって上記ベクターを導入し、形質転換を行った。この形質転換酵母をYPD液体培地で一晩培養した後、0.5 μg/mlのオーレオバシジンＡ含有YPD固体培地に塗布し、生育してきたコロニーについて、導入ベクターがCHO2遺伝子部位に挿入されている事を確認できた。
The mutation was introduced into the CHO2 gene of the yeast genome using the above CHO2 mutagenesis vector. Specifically, laboratory yeast (Saccharomyces cerevisiae BY20592) was used as a parent strain, and the above vector was introduced by the lithium acetate method for transformation. This transformed yeast is cultured overnight in a YPD liquid medium, then applied to a 0.5 μμg / ml aureobasidin A-containing YPD solid medium, and the introduced vector is inserted into the CHO2 gene site for the grown colonies. Was confirmed.
次に、そのコロニーをYP-Galactose固体培地（2w/v％ペプトン、1w/v％イーストエキス、2w/v％ガラクトース、２w/v%精製寒天）に塗布し、28℃で3日間培養した。ここで、pAUR135ベクターにはガラクトース誘導致死性があるため、生育してくるコロニーはベクター配列が脱落した復帰変異体であり、その中にはcho2変異導入ベクターに導入したストップコドンがゲノムに導入された株が含まれる。そこで、CHO2遺伝子にストップコドンが導入されたコロニー（以後、この株を「cho2変異体」という）のゲノムをシークエンスする事により選抜した。
Next, the colonies were spread on a YP-Galactose solid medium (2 w / v% peptone, 1 w / v% yeast extract, 2 w / v% galactose, 2 w / v% purified agar) and cultured at 28 ° C. for 3 days. Here, since the pAUR135 vector has galactose-induced lethality, the growing colony is a revertant in which the vector sequence has been dropped, and the stop codon introduced into the cho2 mutagenesis vector is introduced into the genome. Stocks included. Therefore, selection was performed by sequencing the genome of a colony in which a stop codon was introduced into the CHO2 gene (hereinafter this strain is referred to as “cho2 mutant”).
（２）コリン添加培地によるSAM生産量の測定
前項で得られた実験室酵母のcho2変異体と野生型親株（BY20592）および清酒酵母（Saccharomyces cerevisiae BY2696、協会7号）を下記の条件で培養し、培養物を得た。すなわち、50mlの遠沈管に5mlのSAM発酵用培地（5w/v％グルコース、1w/v％ペプトン、0.5w/v％イーストエキス、0.4w/v％KH2PO4、0.2w/v％K2HPO4、0.05w/v％Mg2SO4・H2O、0.15w/v％L-メチオニン、pH6.0）を分注した後、最終濃度が10μＭ～100ｍＭの範囲となるように10倍濃度毎にコリンを添加した。これらの培地に酵母株をそれぞれ接種し、28℃、72時間浸とう培養を行い、十分量の培養物を得た。 (2) Measurement of SAM production using choline-added medium The laboratory yeast cho2 mutant obtained in the previous section, wild type parent strain (BY20592) and sake yeast (Saccharomyces cerevisiae BY2696, Association No. 7) were cultured under the following conditions. A culture was obtained. That is, 5 ml of SAM fermentation medium (5 w / v% glucose, 1 w / v% peptone, 0.5 w / v% yeast extract, 0.4 w / v% KH 2 PO 4 , 0.2 w / v% K in a 50 ml centrifuge tube 2 HPO 4 , 0.05 w / v% Mg 2 SO 4 .H 2 O, 0.15 w / v% L-methionine, pH 6.0) is dispensed, and the final concentration is 10 μM to 100 mM. Choline was added at every double concentration. Each of these media was inoculated with a yeast strain and immersed in culture at 28 ° C. for 72 hours to obtain a sufficient amount of culture.
前項で得られた実験室酵母のcho2変異体と野生型親株（BY20592）および清酒酵母（Saccharomyces cerevisiae BY2696、協会7号）を下記の条件で培養し、培養物を得た。すなわち、50mlの遠沈管に5mlのSAM発酵用培地（5w/v％グルコース、1w/v％ペプトン、0.5w/v％イーストエキス、0.4w/v％KH2PO4、0.2w/v％K2HPO4、0.05w/v％Mg2SO4・H2O、0.15w/v％L-メチオニン、pH6.0）を分注した後、最終濃度が10μＭ～100ｍＭの範囲となるように10倍濃度毎にコリンを添加した。これらの培地に酵母株をそれぞれ接種し、28℃、72時間浸とう培養を行い、十分量の培養物を得た。 (2) Measurement of SAM production using choline-added medium The laboratory yeast cho2 mutant obtained in the previous section, wild type parent strain (BY20592) and sake yeast (Saccharomyces cerevisiae BY2696, Association No. 7) were cultured under the following conditions. A culture was obtained. That is, 5 ml of SAM fermentation medium (5 w / v% glucose, 1 w / v% peptone, 0.5 w / v% yeast extract, 0.4 w / v% KH 2 PO 4 , 0.2 w / v% K in a 50 ml centrifuge tube 2 HPO 4 , 0.05 w / v% Mg 2 SO 4 .H 2 O, 0.15 w / v% L-methionine, pH 6.0) is dispensed, and the final concentration is 10 μM to 100 mM. Choline was added at every double concentration. Each of these media was inoculated with a yeast strain and immersed in culture at 28 ° C. for 72 hours to obtain a sufficient amount of culture.
上述の72時間培養物に蓄積されるSAMを以下のような方法で抽出し、定量した。すなわち、菌体を遠心にて沈降させ、上清を除いた後、10％過塩素酸を添加してSAMを抽出（室温、1時間）し、その上清をペーパークロマトグラフィー（展開溶媒 EtOH：n-BuOH：水：AcOH：1w/v％NaP２O７＝３０：３５：４０：１：２）にて精製し、SAMスポットを切り出して抽出したものを分析用サンプルとした。
SAM accumulated in the above 72-hour culture was extracted and quantified by the following method. That is, the cells are sedimented by centrifugation, the supernatant is removed, 10% perchloric acid is added to extract SAM (room temperature, 1 hour), and the supernatant is subjected to paper chromatography (developing solvent EtOH: n-BuOH: water: AcOH: 1 w / v% NaP 2 O 7 = 30: 35: 40: 1: 2), and the sample extracted by cutting out the SAM spot was used as an analytical sample.
定量はHPLCを用いて260ｎmのUV吸収を指標に標準品との比較で行った。分析条件としては、使用装置：Waters 2690 Separation Module Waters 2487 Dual Absorbance Detectorシステム、使用カラム：cosmosil packed column 5C18-MS(4.6i.d.×250mm)、溶出溶媒：5v/v％メタノール-95v/v％0.2M KH２PO４溶液、流速：1ml/min、カラム温度：25℃であった。
Quantification was performed by comparison with a standard product using UV absorption of 260 nm as an index using HPLC. Analytical conditions are as follows: Equipment used: Waters 2690 Separation Module Waters 2487 Dual Absorbance Detector system, Column used: cosmosil packed column 5C18-MS (4.6id x 250mm), Elution solvent: 5v / v% Methanol-95v / v% 0.2M KH 2 PO 4 solution, flow rate: 1 ml / min, column temperature: 25 ° C.
その結果、全ての株でコリン添加によるSAMの蓄積量の向上が確認された。さらにその効果は１mＭまで濃度依存的に向上する事が観察され、それ以上の濃度では効果は飽和する事が分かった（図３及び４）。
As a result, it was confirmed that the amount of accumulated SAM by adding choline was improved in all strains. Further, it was observed that the effect was improved in a concentration-dependent manner up to 1 mM, and the effect was saturated at a concentration higher than that (FIGS. 3 and 4).
さらに、コリン添加の効果をcho2変異体とその親株の培養物において比較した。その結果、SAM収量増加効果はcho2変異体を培養した方が効果的であった。これはCHO2遺伝子がホスファチジルコリンの生合成に関与する遺伝子である事からも明らかであり、ホスファチジルコリンのde novoの生合成系の抑制だけではなく、不足していたホスファチジルコリンがコリンの添加によって補給される事による生育改善などが相乗的に効果を及ぼしていると推測される。実際に図５に示すように、コリン添加濃度に応じて生育は改善され、野生型に比べて大きな生育促進効果を示している。この事から、コリン添加は特にcho2変異体によるSAM生産性向上に対して有効であると言える。
実施例で使用したプライマーの配列を第１表に示す。 Furthermore, the effect of choline addition was compared between the cho2 mutant and its parent strain culture. As a result, the SAM yield increase effect was more effective when the cho2 mutant was cultured. This is evident from the fact that the CHO2 gene is involved in the biosynthesis of phosphatidylcholine, not only suppressing the de novo biosynthesis system of phosphatidylcholine, but also supplementing the missing phosphatidylcholine by the addition of choline. It is presumed that the growth improvement by the plant has a synergistic effect. Actually, as shown in FIG. 5, the growth is improved in accordance with the choline addition concentration, indicating a greater growth promoting effect than the wild type. From this, it can be said that the addition of choline is particularly effective for improving the SAM productivity by the cho2 mutant.
Table 1 shows the primer sequences used in the examples.
実施例で使用したプライマーの配列を第１表に示す。 Furthermore, the effect of choline addition was compared between the cho2 mutant and its parent strain culture. As a result, the SAM yield increase effect was more effective when the cho2 mutant was cultured. This is evident from the fact that the CHO2 gene is involved in the biosynthesis of phosphatidylcholine, not only suppressing the de novo biosynthesis system of phosphatidylcholine, but also supplementing the missing phosphatidylcholine by the addition of choline. It is presumed that the growth improvement by the plant has a synergistic effect. Actually, as shown in FIG. 5, the growth is improved in accordance with the choline addition concentration, indicating a greater growth promoting effect than the wild type. From this, it can be said that the addition of choline is particularly effective for improving the SAM productivity by the cho2 mutant.
Table 1 shows the primer sequences used in the examples.
本発明は、ＳＡＭの大量生産を可能とするため、治療薬やサプリメント等を始め、医療分野その他において広く使用されるＳＡＭの大量供給に寄与する。
なお、２００８年２月１２日に出願された日本特許出願２００８－０３０６７２号の明細書、特許請求の範囲、図面及び要約書の全内容をここに引用し、本発明の明細書の開示として、取り入れるものである。 In order to enable mass production of SAM, the present invention contributes to mass supply of SAM widely used in the medical field and others including therapeutic drugs and supplements.
It should be noted that the entire contents of the specification, claims, drawings and abstract of Japanese Patent Application No. 2008-030672, filed on February 12, 2008, are cited herein as disclosure of the specification of the present invention. Incorporated.
なお、２００８年２月１２日に出願された日本特許出願２００８－０３０６７２号の明細書、特許請求の範囲、図面及び要約書の全内容をここに引用し、本発明の明細書の開示として、取り入れるものである。 In order to enable mass production of SAM, the present invention contributes to mass supply of SAM widely used in the medical field and others including therapeutic drugs and supplements.
It should be noted that the entire contents of the specification, claims, drawings and abstract of Japanese Patent Application No. 2008-030672, filed on February 12, 2008, are cited herein as disclosure of the specification of the present invention. Incorporated.
SEQ ID NO: 1, Primer for amplification of CHO2 gene
SEQ ID NO: 2, Primer for amplification of CHO2 gene
SEQ ID NO: 3, Mutagenic primer containing mutations for amplification of CHO2 gene
SEQ ID NO: 4, Mutagenic primer containing mutations for amplification of CHO2 gene SEQ ID NO: 1, Primer for amplification of CHO2 gene
SEQ ID NO: 2, Primer for amplification of CHO2 gene
SEQ ID NO: 3, Mutagenic primer containing mutations for amplification of CHO2 gene
SEQ ID NO: 4, Mutagenic primer containing mutations for amplification of CHO2 gene
SEQ ID NO: 2, Primer for amplification of CHO2 gene
SEQ ID NO: 3, Mutagenic primer containing mutations for amplification of CHO2 gene
SEQ ID NO: 4, Mutagenic primer containing mutations for amplification of CHO2 gene SEQ ID NO: 1, Primer for amplification of CHO2 gene
SEQ ID NO: 2, Primer for amplification of CHO2 gene
SEQ ID NO: 3, Mutagenic primer containing mutations for amplification of CHO2 gene
SEQ ID NO: 4, Mutagenic primer containing mutations for amplification of CHO2 gene
Claims (10)
- コリン系化合物を添加した培地で酵母を培養し、系内に蓄積したＳ－アデノシルメチオニンを回収することを特徴とするＳ－アデノシルメチオニンを生産する方法。 A method for producing S-adenosylmethionine, comprising culturing yeast in a medium containing a choline-based compound and recovering S-adenosylmethionine accumulated in the system.
- 前記コリン系化合物がコリン、ホスファチジルコリン、ホスホコリン、シチジン－５’－ジリン酸コリン、又はアセチルコリンである請求項１に記載の方法。 The method according to claim 1, wherein the choline-based compound is choline, phosphatidylcholine, phosphocholine, cytidine-5'-diphosphate choline, or acetylcholine.
- 前記酵母がサッカロミセス（Saccharomyces）属である請求項１に記載の方法。 The method according to claim 1, wherein the yeast belongs to the genus Saccharomyces.
- 前記酵母がサッカロミセス セレヴィシエ（Saccharomyces cerevisiae）である請求項３に記載の方法。 The method according to claim 3, wherein the yeast is Saccharomyces cerevisiae.
- 前記酵母が、ホスファチジルコリン合成系酵素をコードする遺伝子において、１個またはそれ以上の塩基が置換、欠失、挿入および／または付加した配列を有する変異体である請求項１に記載の方法。 The method according to claim 1, wherein the yeast is a mutant having a sequence in which one or more bases are substituted, deleted, inserted and / or added in a gene encoding a phosphatidylcholine synthase.
- 前記酵母がcho2変異体である請求項５に記載の方法。 The method according to claim 5, wherein the yeast is a cho2 mutant.
- 前記コリン系化合物を添加した培地が、１０μＭ以上のコリン系化合物を含む培地である請求項１に記載の方法。 The method according to claim 1, wherein the medium supplemented with the choline compound is a medium containing 10 μM or more of the choline compound.
- 前記コリン系化合物を添加した培地が、０．１～１００ｍＭのコリン系化合物を含む培地である請求項７に記載の方法。 The method according to claim 7, wherein the medium supplemented with the choline compound is a medium containing 0.1 to 100 mM choline compound.
- 前記コリン系化合物を添加した培地が、メチオニンを含む培地である請求項１に記載の方法。 The method according to claim 1, wherein the medium supplemented with the choline compound is a medium containing methionine.
- 前記コリン系化合物を添加した培地が、０．０１w/v％以上のメチオニンを含む培地である請求項９に記載の方法。 The method according to claim 9, wherein the medium supplemented with the choline compound is a medium containing 0.01 w / v% or more methionine.
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| JP2001112474A (en) * | 1999-10-14 | 2001-04-24 | Kansai Tlo Kk | Variant of saccharomyces cerevisiae |
| JP2005261361A (en) * | 2004-03-19 | 2005-09-29 | Hiroshima Univ | Yeast variant and utilization thereof |
| WO2008020595A1 (en) * | 2006-08-15 | 2008-02-21 | Ishihara Sangyo Kaisha, Ltd. | Novel method for utilization of microbial mutant |
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| JP2001112474A (en) * | 1999-10-14 | 2001-04-24 | Kansai Tlo Kk | Variant of saccharomyces cerevisiae |
| JP2005261361A (en) * | 2004-03-19 | 2005-09-29 | Hiroshima Univ | Yeast variant and utilization thereof |
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