JP5641192B2 - Method for obtaining S-adenosylmethionine highly accumulating yeast - Google Patents

Description

  The present invention relates to a method for obtaining S-adenosylmethionine (hereinafter also referred to as SAMe, SAM) highly accumulating yeast, and the like. In detail, it is related with the screening method by the selective culture medium of SAM high accumulation yeast, and the mass production method of SAM using SAM high accumulation yeast.

  SAM is a key substance for metabolism of sulfur-containing amino acids present in the living body, and is synthesized from methionine and ATP by the action of methionine adenosyltransferase. It has an active methylthioether group in its structure, and functions as a major methyl group donor in vivo by donating a methyl group to various methyl group acceptors such as proteins and nucleic acids. According to medical clinical trials, there are reports that there are physiological functions that exert preventive and ameliorating effects on various diseases such as alcoholic liver dysfunction, arteriosclerosis, senile dementia, depression, and arthritis. In recent years, it has been newly reported that it acts on cancer and AIDS, and has established itself as a prescription drug in the EU countries and a supplement in the United States. In Japan, the Ministry of Health, Labor and Welfare in February 2009 showed that there was no problem in describing SAMe in nutritional labeling etc. for foods such as yeast naturally contained in SAMe (SAM) In the near future, it is a substance that is expected to activate the market.

  From the knowledge to date, it is known that this SAM has a high production capacity in yeast among all microorganisms. Currently, industrial production of SAM is carried out by extraction from Saccharomyces cerevisiae. There are many unclear points about the production mechanism, and the production cost is high due to the need to add a large amount of expensive amino acids to the yeast culture medium, so that the mechanism of action of SAM is elucidated and faster than conventional SAM production systems. The construction of a system that can be mass-produced at low cost is a current problem.

  Conventional SAM production has been attempted to improve productivity by using an existing strain and examining the composition of the production medium of the bacterium. Finally, recently, attempts have been made to obtain mutant strains with further improved productivity from among SAM-producing yeasts, but no significant improvement in SAM productivity has been achieved (Patent Document 1). Further improvements such as requiring a complicated operation such as self-cloning (Patent Document 2), particularly difficult to use as a food, or a genetic recombinant in which a specific gene is deleted by genetic manipulation (Patent Document 3). There is room for.

JP 2004-283103 A JP 2009-034043 A JP 2009-213469 A

  In the present invention, because of the mass production of S-adenosylmethionine (SAM), the experimental operation is simpler than that of a conventional SAM high-producing yeast screening system, and acquisition of a highly productive SAM high-accumulating yeast is achieved. It is an object of the present invention to provide a method and a mass production method of SAM using the yeast obtained thereby.

In order to achieve the above object, the present inventors conducted intensive research, cultured yeast using a selective medium containing cordycepin, and selected a cordycepin-resistant strain that is a mutant yeast that grows (positive screening). ) And found that SAM high-accumulation yeast can be obtained simply and efficiently, and led to the present invention.
It was also found that mass production of SAM can be industrially performed by culturing in a predetermined medium using the obtained yeast.

That is, the embodiment of the present invention is as follows.
(1) Obtaining a cordycepin-resistant strain that grows on a selective medium containing cordycepin, a method for obtaining a yeast that accumulates highly accumulated S-adenosylmethionine (2 to 20 times the amount of the parent strain (for example, 2 to 2) 30 times the amount) a method for obtaining a SAM high-accumulation yeast that highly accumulates SAM.
(2) S-adenosylmethionine highly accumulating yeast, which is a cordycepin-resistant mutant that is not a gene recombinant, obtained by the method according to (1).
(3) The S-adenosylmethionine highly accumulating yeast according to (2), wherein the yeast belongs to the genus Saccharomyces or Candida.
(4) The S-adenosylmethionine highly accumulating yeast according to (3), wherein the yeast is Saccharomyces cerevisiae.
(5) Sake yeast NY9-10 (NITE P-831), which is a S-adenosylmethionine high accumulation yeast having resistance to cordycepin.
(6) Sake yeast NY7-3 (NITE P-830), which is an S-adenosylmethionine high accumulation yeast having resistance to cordycepin.
(7) S- characterized by culturing the yeast according to any one of (2) to (6) and obtaining S-adenosylmethionine from the culture (medium and / or cells). A method for mass production of adenosylmethionine.
(8) An S-adenosylmethionine highly accumulating yeast selective medium characterized by comprising cordycepin.

  According to the present invention, a SAM high accumulation (or SAM high content) yeast can be easily and efficiently obtained by positive screening of a cordycepin resistant strain using a cordycepin-containing selective medium. It is also possible to obtain yeast that accumulates SAM at high concentrations and / or secretes SAM outside the cells. And mass production of SAM can be performed industrially and efficiently by using the obtained yeast.

The SAM accumulation amount when 25 strains (NY9-1 to 25) of the cordycepin resistant strains obtained from the K-9 strain were cultured with shaking in a YPD medium at 30 ° C. for 24 hours (circles indicate the deposited strain (NITE P- 831)). In addition, dry cell weight (DCW) shows a dry cell weight. same as below. The SAM accumulation amount when nine strains (NY7-1 to 9) of the cordycepin resistant strains obtained from the K-7 strain were cultured with shaking in YPD medium at 30 ° C. for 24 hours (circles indicate the deposited strain (NITE P- 830)). The SAM accumulation amount when five strains (NY9-1, 4, 9, 10, 24) of cordycepin resistant strains obtained from the K-9 strain were cultured with shaking in an O-medium at 30 ° C. for 24 hours (circles) Indicates the deposited strain (NITE P-831). The SAM accumulation amount when three strains (NY7-3, 5, 6) obtained from K-7 strain were cultured with shaking in O-medium at 30 ° C. for 24 hours (circles indicate the deposited strain ( NITE P-830)).

  In the present invention, a selection medium containing cordycepin (3-deoxyadenosine) is used. Cordycepin is a derivative of adenosine, a ribonucleoside, with a structure lacking oxygen at the 3-position of the ribose site of adenosine, which inhibits the normal function of adenosine, an intermediate metabolite of the yeast methionine metabolic pathway. . Thus, it is considered that methionine is not normally metabolized and is highly accumulated in the cell as SAM.

  Normal yeast cannot grow on a medium containing cordycepin. In the present invention, a normal yeast (wild strain) may be used as it is, and a method of allowing a resistant strain to appear accidentally may be used. Can be acquired efficiently. A conventional method is appropriately used for the mutation treatment, and either a physical method or a chemical method can be used. Examples of the physical method include ultraviolet irradiation and radiation (for example, γ-ray) irradiation, and examples of the chemical method include nitrous acid, nitrogen mustard, acridine dye, ethyl methanesulfonic acid (EMS), and N-methyl. There is a method of suspending bacterial cells in a solution of a mutation agent such as -N'-nitro-N-nitrosoguanidine (NTG). These methods can also be performed in combination as appropriate.

  In the present invention, a genetically modified yeast deficient in a specific gene is not used. Therefore, in the present invention, it is not a gene recombinant, that is, a mutant strain in which a wild strain is accidentally mutated or a mutant strain that has emerged through the above-described mutation treatment (in the present invention, these are collectively referred to as “mutant strains”). There is no problem even if it is used for food.

  Subsequently, the above-mentioned wild strain yeast or mutation-treated yeast is cultured in a cordycepin-containing medium to obtain a cordycepin resistant strain that grows. Since the screening in the present invention is a positive screening, it is very efficient because the operation is simple and result determination is performed accurately. The addition of cordycepin is exemplified by YPD medium (yeast extract 1%, peptone 2%, glucose 2%) containing 500 to 1000 μg (more preferably 600 to 800 μg) per ml in the case of sake yeast in liquid culture. About other yeast, what is necessary is just to determine suitably based on the said numerical range.

  Examples of yeast include Candida utilis, C.I. Examples include yeast belonging to the genus Candida such as Macedoniensis and the genus Saccharomyces. In the latter, Saccharomyces cerevisiae, S. et al. rouxii, S .; uvarum, S.M. Bayanus, S.B. chevaleri, S .; An example is baili.

  In addition, various commercially available yeasts, practical yeasts, laboratory yeasts and the like belonging to the genus Saccharomyces are also objects of the present invention, and sake yeasts (Association No. 6 yeast, Association No. 7 yeast, Association No. 9 yeast, Association 10). No. yeast etc.), shochu yeast (Japan Brewing Association Shochu No. 2 yeast, etc.), wine yeast (vinegar No. 1 yeast (Japan Brewing Association No. 1 yeast), wine No. 3 yeast, wine No. 4 yeast etc. ), Brewer's yeast, baker's yeast and the like are also given as non-limiting examples.

  In the present invention, the target SAM high accumulation yeast can be obtained by the method as described above. As an example, Saccharomyces cerevisiae K-9 (Association No. 9 yeast) and K-7 (Association No. 7 yeast), which are sake yeasts, were successfully obtained as parent strains, respectively, The strain acquired from K-9 was named NY9-10, the strain acquired from K-7 was named NY7-3, and NITE P-831 and NITE P-830 were assigned to the Patent Microorganism Depositary, National Institute of Technology and Evaluation. As deposited.

A large amount of SAM can be obtained from the culture by culturing the SAM high-accumulation strain separated and bred in this manner. In the YPD medium, SAM can be accumulated 20 times more than the parent strain in the same medium, and if it is desired to produce and accumulate in large quantities, for example, O-medium (glucose 5%, peptone 1%, yeast extract 0 5%, KH ₂ PO ₄ 0.4%, K ₂ HPO ₄ 0.2%, MgSO ₄ .7H ₂ O 0.05%, L-methionine 0.15%) The mutant strain selected from 1 has a high accumulation of 1 to 80 mg (for example, 3 to 80 mg), sometimes 80 mg or more of SAM per 1 g of yeast cell dry matter weight, and the possibility of accumulating 100 mg or more is fully recognized. In addition, according to the present Example, accumulation | storage of about 2-20 times SAM of a parent strain was confirmed, However Accumulation beyond that (for example, 10-30 times) is also possible enough.

  As described above, a large amount of SAM can be obtained by culturing SAM high-accumulation yeast (cordycepin-resistant strain) and growing the yeast, and extracting and obtaining SAM from the grown yeast culture. . Culture may be carried out according to a conventional method, in which case there is no particular difference in the growth characteristics between the parent strain and the mutant strain (the mutant strain may show better growth than the parent strain). Seen).

In addition, although there is no problem even if YPD medium is used for the culture, it is more preferable to use a methionine-added medium (O-medium etc.), and further mass acquisition of SAM becomes possible. The amount of methionine added may be appropriate, but is preferably in the range of 0.15% to 0.75%.
Furthermore, it is more preferable to use at least one additional medium selected from phosphate, sulfate, and ethanol. These addition amounts may be appropriate, but are preferably about 0.2 to 0.4%, 0.05%, and 4.0%, respectively.

  Examples of the present invention will be described below.

  Saccharomyces cerevisiae K-9 (Association No. 9 yeast) and K-7 (Association No. 7 yeast) were used as parent strains, and mutation treatment was performed by ultraviolet irradiation based on a conventional method. The survival rate was approximately 10%. The treated strain was inoculated in a YPD liquid medium containing 600 μg / ml of cordycepin, and the K-9 strain was turbid after 3 times of culture at 30 ° C. for 3 days and 1 day of culture for 3 times. The bacterial cells were diluted to the fifth power of 10, and 100 ml was applied to a YPD plate and cultured at 30 ° C. The grown colonies were picked up and 260 strains of cordycepin resistant strains were selected and obtained. For the K-7 strain, subculture was repeated 5 times for 3 days at 30 ° C. and 3 times for 1 day, and the turbid cells were diluted to the fifth power of 10 and 100 ml was applied to the YPD plate. Cultivation was carried out at 30 ° C., colonies that had grown were picked up, and 93 strains resistant to cordycepin were selected and obtained.

  Thus, 25 strains (NY9-1 to 25) from strains derived from K-9 bacteria and 9 strains (NY7-1) from strains derived from K-7 bacteria were randomly selected from the strains selected as resistant strains of cordycepin. -9) was selected, pre-cultured in YPD medium at 30 ° C for 24 hours, and cultured in YPD medium at 30 ° C for 24 hours with shaking. SAM was extracted from the cultured cells using 10% perchloric acid (30 ° C., 1 hour), the supernatant was diluted, and the amount of SAM accumulated was analyzed by capillary electrophoresis.

SAM analysis was measured by capillary electrophoresis in the following manner. The analysis conditions are as follows.
Analyzer capillary electrophoresis system (Agilent, 1100 series)
Analysis conditions Capillary: HPCE stndrd cap 75 μm × 72 cm
Buffer: 40 mM sodium phosphate buffer (pH 2.5)
Voltage: positive, 20kV
Sampling: 4 seconds, hydrostatic temperature: 25 ° C
Detection: UV254nm

  Fig. 1 shows the amount of SAM in the cells of 25 strains (NY9-1 to 25) acquired from the K-9 strain, and 9 strains (NY7-1 to 9) acquired from the K-7 strain in Fig. 2 together with the parent strain. It was. As a result, in K-9 strain, 25 out of 25 cordycepin resistant strains were about 20 times (8-12 mg SAM / g DCW), and in K-7 strain, about 9 out of 9 out of 9 cordycepin resistant strains. (About 3 mg SAM / g DCW), SAM was significantly accumulated from the parent strain.

  Furthermore, 5 strains (NY9-1, 4, 9, 10, 24) from K-9 strains and 3 strains from K-7 strains were selected from among the strains selected as cordycepin resistant strains ( NY7-3, 5, 6) was selected, pre-cultured in YPD medium at 30 ° C. for 24 hours, and cultured in O-medium at 30 ° C. for 24 hours with shaking. SAM was extracted from the cultured cells using 10% perchloric acid (30 ° C., 1 hour), the supernatant was diluted, and the amount of SAM accumulated was analyzed by capillary electrophoresis.

  FIG. 3 shows 5 strains (NY9-1, 4, 9, 10, 24) acquired from the K-9 strain, and FIG. 4 shows 3 strains (NY7-3, 5, 6) acquired from the K-7 strain. The amount of SAM in the body was shown together with the parent strain. As a result, in the K-9 strain, about 5 out of 5 cordycepin resistant strains were about 4 times (about 80 mg SAM / g DCW), and in the K-7 strain about 3 times out of 3 out of 3 cordycepin resistant strains ( 60-70 mg SAM / g DCW), SAM was significantly accumulated from the parent strain.

  The present invention is summarized as follows.

  In the present invention, because of the mass production of S-adenosylmethionine (SAM), the experimental operation is simpler than that of a conventional SAM high-producing yeast screening system, and acquisition of a highly productive SAM high-accumulating yeast is achieved. It is an object of the present invention to provide a method, and to provide a method for mass production of SAM using yeast obtained thereby.

  Then, by culturing yeast using a selective medium containing cordycepin and selecting a growing yeast (cordycepin resistant strain), it is possible to efficiently obtain SAM highly accumulating yeast. And mass production of SAM can be industrially performed by culturing with a predetermined culture medium using obtained yeast.

The accession numbers of the microorganisms deposited in the present invention are shown below.
(1) Sake yeast NY9-10 (NITE P-831).
(2) Sake yeast NY7-3 (NITE P-830).

Claims (5)

Obtaining a cordycepin-resistant strain that grows in a selective medium containing 500 to 1000 μg of cordycepin per ml, and selecting one that accumulates 1 to 80 mg of S-adenosylmethionine per gram of yeast cell dry matter weight ; A method for obtaining a yeast having a high S-adenosylmethionine accumulation. The method according to claim 1, wherein yeast belonging to the genus Saccharomyces is used as a parent strain. S-adenosylmethionine highly accumulating yeast Sake yeast NY9-10 (NITE P-831). S-adenosylmethionine highly accumulating yeast Sake yeast NY7-3 (NITE P-830).     A method for mass production of S-adenosylmethionine, comprising culturing the yeast according to claim 3 or 4 and obtaining S-adenosylmethionine from the culture.

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