JP6172814B2 - Method for producing yeast exhibiting high ribonucleic acid yield - Google Patents

Description

  The present invention relates to a method for producing yeast exhibiting a high ribonucleic acid yield.

  RNA (ribonucleic acid) is used as a raw material for umami seasonings, functional foods and pharmaceuticals, and technology for efficiently producing RNA is industrially important.

  In order to produce RNA, a method is generally used in which yeast is cultured in a medium containing a carbon source, a nitrogen source, and a phosphorus source, and RNA is extracted from yeast cells. There have been many attempts to efficiently produce RNA, such as a method for increasing the RNA content of yeast cells and a method for efficiently extracting RNA in the yeast cells obtained.

As an example of the former, breeding of strains is mainly carried out practically, and a method of obtaining a mutant strain sensitive to potassium chloride (Non-patent Document 1), the growth is significantly inhibited in a low-temperature environment. A method for searching for mutants (Patent Document 1), a method for searching for mutants having resistance to thiazine / oxazine dyes and acridine dyes (Patent Document 2), Examples thereof include a method for reintroducing the Rrn10 gene (Patent Document 3) and a method for obtaining a strain lacking the FOB1 gene (Patent Document 4).

Examples of the latter include a method of heating yeast cells together with a surfactant (Patent Document 5), extraction in an alkaline solution such as caustic soda, or the presence of a salt produced by pretreatment with alkali and neutralization with an acid. As a method for obtaining low-cost and high-quality RNA, a cell treated at 40 to 60 ° C. under mildly acidic conditions is known. There is a method of heating, extracting at 95 ° C. by adding salt water (Patent Document 7), and this method is often adopted in industrial scale production.
However, RNA extraction from yeast cells has a drawback that the extraction yield varies greatly depending on the type of strain used, medium components, and the state of yeast cells.

On the other hand, as an industrial yeast culture method for obtaining RNA, batch culture, fed-batch culture, continuous culture, and the like are used, but continuous culture is often selected from the viewpoint of cost and equipment scale. . Therefore, obtaining a yeast strain that can stably obtain RNA at a high yield in continuous culture is considered important for stable industrial production of RNA. The acquisition method was not known.

JP-B 56-46824 JP-B 48-32350 JP 2009-50247 A JP 2007-75013 A No.45-33657 Japanese Examined Sho 38-8140 Japanese Patent Publication No.52-18200

Doi et al., Abstracts of Nikko Kagaku Lecture, P347

  Therefore, an object of the present invention is to establish a method for obtaining a yeast having a high RNA yield, particularly a yeast strain suitable for industrial RNA production.

In order to solve the above-mentioned problems, the inventors have studied a method for producing RNA using yeast, and by selecting yeast mutants having resistance to macrolide antibiotics, a high RNA yield can be obtained. It has been found that the strains shown can be efficiently obtained, and that the obtained mutant strains have a particularly high RNA yield in continuous culture, and the present invention has been completed.

According to the method of the present invention, a yeast mutant exhibiting a higher RNA yield than the parent strain can be efficiently obtained. In particular, a strain exhibiting a high RNA yield obtained by the method of the present invention exhibits a very high RNA yield when used in continuous culture, and thus is a strain that is extremely suitable for industrial RNA production.

  In this specification, RNA yield means the value (g / L) which showed the amount of extracted RNA by the weight concentration per original culture solution.

  The yeast mutant of the present invention shows an RNA yield of 1.05 to 3 times that of the parent strain, and particularly 1.2 to 3 times that of the parent strain when used in continuous culture. For this reason, it is a very suitable mutant for industrial RNA production.

  As the yeast, any one that can be usually used for the production of foods and pharmaceutical raw materials can be used, and examples include yeast belonging to the genus Candida, Saccharomyces, Pichia, Schizosaccharomyces, and the like. Further specific examples include Candida utilis and Candida tropicalis as examples of the genus Candida, Saccharomyces cerevisiae as an example of the genus Saccharomyces, and Pichia as an example of the Pichia genus As examples of the genus Pichia pastoris and Schizosaccharomyces, there can be mentioned Schizosaccharomyces pombe. Among these, yeast belonging to the genus Candida which is often used for the production of RNA is preferable.

  The present invention includes (1) a step of treating a yeast with a mutagen, (2) a step of selecting a yeast mutant having resistance to macrolide antibiotics, and (3) an RNA yield higher than that of the parent strain among the selected strains. The present invention relates to a method for obtaining a yeast mutant strain having a high RNA yield, comprising a step of obtaining a strain having a high strain.

  The mutagen treatment method of (1) is not particularly limited, but a known method such as a physical method using ultraviolet rays or ionizing radiation or a chemical method using nitrous acid, nitrosoguanidine, or methyl methanesulfonate is used. Use it.

  As a method for selecting mutant strains having resistance to macrolide antibiotics in (2), for example, yeast is seeded on an agar medium containing 0.02 ppm or more of macrolide antibiotics, and surviving strains are selected. do it. The macrolide antibiotic is an antibiotic having a macrocyclic lactone, and examples thereof include rapamycin, reinamycin, lankacidin C, tacrolimus (FK506) and the like.

  In addition, process (1) and (2) can also be implemented in order of (1) and (2), or can also be performed simultaneously. (1) When performing (2) at the same time, inoculate the parent strain of yeast on an agar medium containing a macrolide antibiotic and irradiate the parent strain on the agar medium with ultraviolet irradiation, etc. Can be done. Then, the mutant which has a macrolide type antibiotic resistance can be selected by selecting the strain | stump | stock which forms a colony on a culture medium.

  As a method for obtaining a strain with a high RNA extraction yield in (3), for example, a plurality of resistant mutant strains are cultured in a small scale in a flask, RNA is extracted from yeast cells by a predetermined method, and a method such as HPLC is used. RNA may be quantified and a strain having a higher yield than the parent strain may be selected.

  According to the method of the present invention, a method of simply performing a mutagen treatment on yeast and selecting a strain exhibiting a high RNA yield, or a method for obtaining a known strain exhibiting a high RNA content (for example, a potassium chloride sensitive strain) Compared with the acquisition method (Non-patent Document 1)), a strain exhibiting a high RNA yield can be obtained very efficiently. Moreover, the mutant strain obtained by the method of the present invention not only has a high RNA yield, but also exhibits a higher RNA yield when used in continuous culture.

  The present invention further provides a method for producing RNA by aerobically cultivating the obtained yeast mutant and extracting RNA from the cultured yeast. In RNA production, the macrolide antibiotic-resistant mutant obtained by the above-described method may be aerobically cultured in a medium containing a carbon source, a nitrogen source, an inorganic salt, and the like.

  As a medium composition for culturing the strain, one or more selected from the group consisting of glucose, sucrose, acetic acid, ethanol, molasses, sulfite pulp waste liquid, etc., which are used as a carbon source for culturing ordinary microorganisms, Used as the nitrogen source is one or more selected from the group consisting of nitric acid and its salts, urea, ammonia and its salts, and nitrogen-containing organic substances such as corn steep liquor, casein, yeast extract or peptone Is done. Further, a phosphate component, a potassium component, and a magnesium component may be added to the medium. These include monoammonium phosphate, phosphoric acid, lime perphosphate, potassium hydroxide, potassium chloride, magnesium chloride, magnesium sulfate, etc. Industrial raw materials may be used. In addition, inorganic ions such as zinc, copper, manganese, and iron ions may be added. Furthermore, vitamins, nucleic acid-related substances and the like may be added.

  The culture format may be batch culture, fed-batch culture or continuous culture. Above all, continuous culture is preferable because RNA can be obtained more efficiently due to the characteristics of the yeast of the present invention.

  The culture temperature may be according to general yeast culture conditions, for example, 20 ° C to 40 ° C, preferably 25 ° C to 35 ° C, and the pH is 2.5 to 8.0, preferably 2.8 to 6 .0 is good.

  The RNA extraction method may be a known method. For example, the culture solution is centrifuged to concentrate the bacterial cells, and then heated under acidic conditions such as hydrochloric acid. Thereafter, the supernatant is removed, water is added to the precipitate to prepare a concentrated slurry, neutralized with alkali, and then a salt such as sodium chloride is added and heat-treated. An RNA extract can be obtained by subjecting the obtained heat-treated product to centrifugation and collecting the supernatant.

  Hereinafter, the present invention will be described with reference to examples. The present invention is not limited by these examples.

(Example 1) Acquisition of mutant strains As parent strains, Candida utilis NBRC0988 strain deposited with Product Evaluation Technology Infrastructure and M25-150 strain which is a Yamasa soy sauce-containing strain were used. The parent strain was cultured overnight in a test tube containing YM medium (0.6% yeast extract, 0.6% malt extract, 1% bactotryptone, 2% glucose, 2% agar). The cultured cells were collected, seeded on an agar medium containing 2 ppm rapamycin and containing glucose as the sole carbon source, and a lethality of 70 − was obtained by ultraviolet irradiation (UV lamp: Panasonic GL-15, wavelength 253.7 nm). Mutation treatment was performed under the condition of 80%. The mutation-treated agar medium was cultured at 30 ° C. for 3 days and nights, and colony formation of resistant strains was confirmed.

480 resistant strains obtained from the NBRC0988 strain strain and 1040 resistant strains obtained from the M25-150 strain strain were cultured on a small scale, and the resulting cells were cultured at pH 2 to 3.5 in the presence of HCl. By selecting a strain having an RNA content higher than that of the parent strain after pretreatment at 60 ° C. for 10 minutes and heating at 95 ° C. for 4 hours, NR-7, NR from the NBRC0988 strain line Three strains of OR-21 and OR-40 were obtained from -21 and NR-22 strains and M25-150 strain, respectively.

(Example 2) Comparison of RNA production and yield in batch culture The parent strains NBRC0988 strain, M25-150 strain and 6 mutant strains obtained in Example 1 were seeded in advance in a flask, and then 3L fermenter A batch culture was performed after inoculation.
Medium composition: 4.2% glucose, 0.2% potassium chloride, 600 ppm magnesium sulfate heptahydrate, 8 ppm iron (III) chloride hexahydrate, 6 ppm manganese chloride tetrahydrate, 0.5 ppm copper sulfate -Pentahydrate, 10 ppm zinc sulfate, heptahydrate, 2.2 g / L monoammonium phosphate, 5 g / L ammonium sulfate, 0.025% Adekanol LG-109. The culture conditions are 1.5 L of medium solution, 30 ° C. temperature, 1000 rpm stirring speed, 1.3 vvm aeration, and oxygen gas is vented depending on the situation so that the dissolved oxygen amount does not fall below 0.2 mg / L. did. Further, 14% ammonia water was added dropwise so as to maintain the pH of 3.2 or higher to maintain the pH.

The obtained culture solution is concentrated by centrifugation so that the dry cell weight becomes 10 to 15%, and then heated to 60 ° C., and hydrochloric acid is added so that the pH becomes 2.0 to 3.5. Then, heat treatment was performed for 10 minutes. Thereafter, the supernatant of centrifugation was removed, and water was added to the precipitate to prepare a suspension in an amount of 15% of the original culture solution. The suspension was neutralized with an aqueous sodium chloride solution, sodium chloride was added to 4%, and the mixture was heated at 95 ° C. for 4 hours. The obtained heat-treated product was subjected to centrifugation, and the supernatant was collected. The precipitate was resuspended by adding an amount of water equal to that of the heat treatment solution, and centrifuged again to recover the supernatant. The collected supernatants were combined and used as an RNA extract.

The obtained RNA extract was appropriately diluted and subjected to gel infiltration high pressure liquid chromatography (hereinafter GPC-HPLC). TSKgel G3000PW _XL was used as the HPLC column, and a solution containing 7M urea and 50 mM Tris-HCl (pH 7.5) was used as the moving bed, and a 260 nm ultraviolet light absorbing substance in the polymer region was detected. The method of Schmitt Tanhauser Schneider [J. Biol. Chem. 1946, 164, 747] (hereinafter referred to as STS method) was subjected to the same analysis, and the RNA yield in the extract was quantified from the ultraviolet absorption peak area.

The RNA yield in the extract obtained from each strain culture solution was as shown in Tables 1 and 2. Three strains obtained from NBRC0988 strain according to the present invention showed stable and high RNA yield. The M25-150 strain is a strain that provides a relatively stable and higher RNA yield than the NBRC0988 strain. However, the two strains obtained from the M25-150 strain according to the present invention have higher yields of RNA. Extraction was possible.

(Example 3) Comparison with known screening method The method of the present invention and a method for obtaining a potassium chloride (KCl) sensitive strain known as a method for obtaining a strain having a high RNA content (hereinafter referred to as "KCl method") Each of which may be compared).
When performing the method of obtaining a KCl sensitive strain, Candida utilis NBRC0988 strain was used as a parent strain. The parent strain was cultured overnight in a test tube containing YM medium (0.6% yeast extract, 0.6% malt extract, 1% bactotryptone, 2% glucose, 2% agar). The cultured cells are collected and seeded on an agar medium containing glucose as the only carbon source, and then the lethality is 70-80% by UV irradiation (UV lamp: Panasonic GL-15, wavelength 253.7 nm). Mutation treatment was performed under such conditions.

  A mutant colony obtained by UV irradiation treatment was collected, and the strain was grown on the agar medium described above and an agar medium supplemented with 8% KCl, and a strain showing good growth only in a medium not containing KCl. By the method of selection, 680 KCl sensitive strains were obtained. Among them, when searching for a mutant strain having an RNA yield of 10% or more higher than that of the parent strain NBRC0988, the strain with the highest RNA yield had an RNA yield of 1.08 times that of the parent strain. No mutant strain with improved RNA yield could be found.

  On the other hand, in the method of the present invention, as described in Examples 1 and 2, among the 480 rapamycin resistant strains obtained from the NBRC0988 strain as a parent strain, a strain having an RNA yield improved by 10% or more than the parent strain was obtained. Three strains were obtained (see Example 2 and Table 1).

  From the above results, it is clear that the method of the present invention is a method capable of efficiently obtaining strains with greatly improved RNA yields compared to the KCl method, which is a known method for obtaining strains containing high RNA. It was.

(Example 4) Comparison of RNA production and yield in continuous culture For the yeast mutant obtained by the method of the present invention and its parent strain, the RNA yield when used in continuous culture was examined.
The NBRC0988 strain, NR-7, NR-22, M25-150, OR-39 and OR-40 were seeded in advance in a flask as the mutant strain obtained in Example 1 and its parent strain, and then a 3L fermenter. The cells were inoculated and continuously cultured. Medium composition is 5.25% glucose, 0.2% potassium chloride, 600 ppm magnesium sulfate heptahydrate, 8 ppm iron (III) chloride hexahydrate, 6 ppm manganese chloride tetrahydrate, 0.5 ppm sulfuric acid Copper pentahydrate, 10 ppm zinc sulfate.7 hydrate, 2.2 g / L monoammonium phosphate. The culture conditions are 1.5 L of medium solution, 30 ° C. temperature, 1000 rpm stirring speed, 1.3 vvm aeration, and oxygen gas is vented depending on the situation so that the dissolved oxygen amount does not fall below 0.2 mg / L. did. During the cultivation, 14% ammonia water was dropped to maintain pH of 3.2 or more to maintain pH. The flow rate of continuous culture was as follows: NBRC0988 strain and its mutant strains NR-7 and NR-22, dilution rate D = 0.36 h ⁻¹ , M25-150 strain and its mutant strains OR-39, OR In -40, it was carried out at D = 0.28 h- ¹ .

The culture broth was collected from the liquid outlet, and the RNA yield per culture broth was determined according to the method of Example 2, and the results were as shown in Tables 3 and 4. As a result, in the parent strains NBRC0988 and M25-150, there was no significant difference in RNA yield between batch culture and continuous culture, whereas in the mutant strain, RNA yield was particularly increased in continuous culture.

On the other hand, among the mutant strains obtained by the KCl method and having the highest RNA yield (the RNA yield in batch culture was 1.08 times that of the parent strain), the RNA yield when continuously cultured was similarly examined. As a result, the RNA yield of the mutant strain during continuous culture was almost the same as that of the parent strain NBRC0988 (about 1.0 times).
Therefore, in the mutant strain obtained by the KCl method, the RNA yield was not particularly increased when used for continuous culture.

From the above results, all mutant strains obtained by the method of the present invention not only have a higher RNA yield than the parent strain, regardless of whether NBRC0988 or M25-150 is used as the parent strain, In particular, it has been clarified that the strain has a very high RNA yield during continuous culture and has a very suitable property for practical production of RNA.

Claims (3)

The method of acquiring the yeast mutant which shows the RNA yield of 1.2 times -3.0 times compared with a parent strain, when used for continuous culture by the acquisition method containing the process of following (1)-(3).
(1) A step of performing a mutagen treatment on yeast,
(2) a step of selecting a mutant having rapamycin resistance from among the yeast strains treated with mutagen,
(3) A step of obtaining a strain having a higher RNA yield than the parent strain from among the selected strains. The acquisition method according to claim 1, wherein the yeast belongs to the genus Candida. A method for producing RNA by continuously aerobically culturing a yeast mutant obtained by the method according to claim 1 or 2 and extracting RNA from the cultured yeast.