JP6886191B2 - Yeast showing high yield of ribonucleic acid - Google Patents

Description

The present invention relates to yeasts that exhibit high yields of ribonucleic acid.

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

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

As an example of the former, in practice, strains are mainly bred, and a method for obtaining a mutant strain resistant to rapamycin (Patent Document 1) and a method for obtaining a mutant strain sensitive to potassium chloride. (Non-Patent Document 1), a method for searching for a mutant strain whose growth is significantly inhibited in a low temperature environment (Patent Document 2), a method for searching for a mutant strain having resistance to a thiazine / oxazine dye and an acrydin dye (Patent Document 2). Patent Document 3), a method of reintroducing the Rrn10 gene into a strain that grows well with an Rrn10-deficient strain (Patent Document 4), a method of obtaining a strain lacking the FOB1 gene (Patent Document 5), and the like can be mentioned.

The latter includes a method of heating yeast cells together with a surfactant (Patent Document 6), extraction in an alkaline solution such as caustic soda, or pretreatment with alkali and neutralization with acid in the presence of a salt produced. (Patent Document 7) and the like are known, but as a method for obtaining high-quality RNA at a low cost, cells treated at 40 to 60 ° C. under weakly acidic conditions are neutralized. A method of adding salt water to the mixture and heating and extracting at 95 ° C. is mentioned (Patent Document 8), and this method is often adopted in industrial scale production.

JP-A-2016-214104 Tokukousho 56-46824 Tokukousho No. 48-32350 JP-A-2009-50247 JP-A-2007-75013 Tokukousho No. 45-33657 Tokukousho 38-8140 Tokukousho 52-18200

Doi et al., Abstract of Nihon Agricultural Chemistry, P347 (1974)

In the above-mentioned known strain breeding method, there is a problem that the selection efficiency of yeast showing high RNA yield is not sufficient as compared with the parent strain, and a great deal of labor and time are required to obtain the target strain. It was.
Therefore, an object of the present invention is to establish and establish an efficient selection method for yeast showing high RNA yield.

In order to solve the above problems, the inventors are studying a method for producing RNA using yeast, and by selecting a yeast mutant strain having vanillin resistance, a strain showing a high RNA yield is efficiently produced. We have found that it can be obtained as a target, and have completed the present invention.

According to the method of the present invention, a yeast mutant strain showing a higher RNA yield than the parent strain can be obtained with extremely high efficiency as compared with the existing mutant strain acquisition method. Furthermore, the strain exhibiting high RNA yield obtained by the present invention is suitable for industrial RNA production because it stably exhibits high RNA yield even when the scale of culture is increased.

In the present specification, the RNA yield refers to a value (g / L) indicating the amount of extracted RNA in terms of weight concentration per original culture medium.

Examples of yeast include Candida yeast that can be usually used in the production of food and pharmaceutical raw materials. As a further specific example, examples of the genus Candida include Candida utilis and Candida tropicalis.

As the parent strain used in the present invention, in addition to the donor strain and a general wild strain, a strain having a high RNA yield obtained by a known breeding method can be used. As a known breeding method, as described above, a method for obtaining a mutant strain resistant to rapamycin, a method for obtaining a mutant strain sensitive to potassium chloride, and a mutant strain whose growth is significantly inhibited in a low temperature environment are searched for. Method, method for searching for mutant strains resistant to thiazine / oxazine dyes, and aclysine dyes, method for reintroducing the Rrn10 gene into a strain that grows well with an Rrn10-deficient strain, and a strain lacking the FOB1 gene. The method of obtaining, etc. can be mentioned, but of course, the method is not limited to these.
The parent strain may be a strain obtained by using one of the above breeding methods, or may be a strain that has undergone a plurality of selections by combining a plurality of methods.

In the present invention, a strain having a higher RNA yield than the parent strain is selected from (1) a step of subjecting yeast to a mutagen treatment, (2) a step of selecting a yeast mutant strain having vanillin resistance, and (3) a selected strain. It relates to a yeast mutant of the genus Candida obtained by a method for obtaining a yeast mutant showing a high RNA yield, which comprises a step of obtaining.
The yeast mutant strain showing a high RNA yield is a mutant strain having an RNA yield of 1.10 times or more as compared with the parent strain.

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

As a method for selecting a mutant strain having resistance to vanillin in (2), yeast may be sown on an agar medium containing vanillin and a surviving strain may be selected. The concentration of vanillin may be 500 ppm or more and 2000 ppm or less, preferably 750 ppm or more and 1500 ppm or less, and more preferably 1000 ppm or more and 1500 ppm or less.

Steps (1) and (2) can be carried out in the order of (1) and (2), or can be carried out at the same time. When performing (1) and (2) at the same time, the parent strain of yeast may be sown on an agar medium containing vanillin, and the parent strain on the agar medium may be irradiated with ultraviolet rays to perform mutagen treatment. .. Then, by selecting a strain that forms a colony on the medium, a mutant strain having vanillin resistance can be selected.

The method for obtaining the strain having a high RNA yield in (3) is not particularly limited, but for example, a vanillin-resistant mutant strain is cultured on a small scale in a flask, RNA is extracted from yeast cells by a predetermined method, HPLC or the like. A strain having a higher RNA yield than the parent strain can be selected by quantifying RNA by the above method.

As for the method of extracting RNA, a known method may be followed. For example, the culture solution is centrifuged to concentrate the cells, and then heat-treated under acidic conditions such as hydrochloric acid. Then, 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 for heat treatment. An RNA extract can be obtained by subjecting the obtained heat-treated product to centrifugation and collecting the supernatant.

The amount of RNA in the obtained RNA extract can be quantified by a known method such as HPLC.
The RNA yield can be calculated by quantifying the amount of RNA and expressing it by the weight concentration per original culture medium.

According to this method, a method of simply performing mutagen treatment on yeast and selecting a strain showing a high RNA yield, or a method of obtaining a strain showing a known high RNA content (for example, a method of obtaining a rapamycin-resistant strain). Compared with (Patent Document 1)), it is possible to obtain a strain showing a high RNA yield extremely efficiently.

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

As the medium composition for culturing the strain, one or more selected from the group consisting of glucose, corn steep liquor, acetic acid, ethanol, sugar honey, sulfite pulp waste liquid, etc., which are used for culturing ordinary microorganisms as a carbon source, are used. As the nitrogen source, one or more selected from the group consisting of nitrate and its salt, urea, ammonia, and its salt, and nitrogen-containing organic substances such as corn steep liquor, casein, yeast extract, and peptone are used. Will be done. Further, a phosphoric acid component, a potassium component, and a magnesium component may be added to the medium, such as monoammonium phosphate, phosphoric acid, lime superphosphate, potassium hydroxide, potassium chloride, magnesium chloride, magnesium sulfate, and the like. It may be an industrial raw material. In addition, inorganic ions such as zinc, copper, manganese, and iron ions may be added. Further, vitamins, nucleic acid-related substances and the like may be added.

The culture form is not particularly limited, and may be any of batch culture, fed-batch culture, and continuous culture.
As the culturing apparatus, known ones can be used without limitation, but when RNA is produced industrially, it is preferable to cultivate using a fermenter because of its scale and convenience.

The culturing temperature may be in accordance with general yeast culturing conditions, for example, 20 to 40 ° C., preferably 25 to 35 ° C., and the pH is 2.5 to 8.0, preferably 2.8 to 6.0. Is good.

The present invention will be described below with reference to examples. The present invention is not limited to these examples.

(Example 1) Acquisition of mutant strain using vanillin resistance as an index Using the Candida Utilis NBRC0988 strain as a parent strain, a mutant strain was acquired by the following method.
The parent strain was cultured day and night in a flask containing YM medium (0.3% yeast extract, 0.3% malt extract, 0.5% casein peptone, 1% glucose, 2% agar). The cultured cells are collected, seeded on an agar medium containing 1000 ppm or 1500 ppm of vanillin and using glucose as the sole carbon source, and subjected to ultraviolet irradiation (UV lamp: Panasonic GL-15, wavelength 253.7 nm) to cause lethality. Mutation treatment was performed under conditions of 70-80%. The mutant-treated agar medium was cultured in a 1500 ppm vanillin-containing medium at 30 ° C. for 3 to 7 days and nights, and colony formation of resistant strains was confirmed.

100 resistant strains obtained from the NBRC0988 strain were cultured in a 500 mL flask containing 40 mL of YM medium, and the obtained bacterial cell culture solution yeast was cultured to obtain a dry cell weight of 10 from the culture solution. After concentrating the cells by centrifugation to a concentration of ~ 15%, the cells were heated to 50 ± 10 ° C., and hydrochloric acid was added to a pH of 2.0 to 3.5 for heat treatment. Centrifugation supernatant was removed and water was added to the precipitate to prepare a turbid solution in 15% of the original culture solution. A 30% aqueous sodium hydroxide solution was added to the turbid solution to neutralize the pH to 6.5 to 7.0, salt was added to a concentration of 4%, and the mixture was heat-treated at 90 ± 5 ° C. The obtained heat-treated product was subjected to centrifugation and the supernatant was collected. The precipitate was resuspended by adding an equal amount of water to the heat treatment solution, and subjected to centrifugation again to recover the supernatant. The RNA extract was obtained by combining the collected supernatants.

The obtained RNA extract was appropriately diluted and subjected to gel infiltration high performance liquid chromatography (hereinafter referred to as GPC-HPLC). _{TSKgel G3000PW XL} was used as the HPLC column, and a 7M urea / 50 mM Tris-HCl (pH 7.5) solution was used as the moving layer. A 260 nm ultraviolet light absorbing substance in the polymer region is detected. Schmidt Tannhäuser Schneider's method [J. Biol. Chem. The RNA solution quantified by 1946, 164, 747] (hereinafter referred to as the STS method) was subjected to the same analysis, and the amount of RNA in the extract was quantified from the area of the ultraviolet absorption peak.
The RNA yield of the obtained mutant strain was calculated by the above method, and a strain having a higher RNA yield than the parent strain was selected.

As a result of this Example 1, 14 strains whose yield was improved by 1.10 times or more were acquired from the parent strain NBRC0988.

(Example 2) Comparison with a known screening method The method of the present invention was compared with a known method for obtaining a mutant strain showing high RNA yield. The KCl method and the rapamycin method were selected as comparison targets.
For the KCl method and the rapamycin method, mutant strains were obtained according to the methods described in Non-Patent Document 1 or Patent Document 1, respectively. The KCl method obtained 680 KCl-resistant mutants, and the rapamycin method obtained 560 rapamycin-resistant mutants.

Each resistant mutant strain obtained from the NBRC0988 strain was cultured in a 500 mL flask containing 40 mL of YM medium, and the obtained bacterial cell culture solution yeast was cultured to obtain a culture solution having a dry cell weight of 10 to 10. After concentrating the cells by centrifugation to 15%, the cells were heated to 50 ± 10 ° C., and hydrochloric acid was added to adjust the pH to 2.0 to 3.5 for heat treatment. Centrifugation supernatant was removed and water was added to the precipitate to prepare a turbid solution in 15% of the original culture solution. A 30% aqueous sodium hydroxide solution was added to the turbid solution to neutralize the pH to 6.5 to 7.0, salt was added to a concentration of 4%, and the mixture was heat-treated at 90 ± 5 ° C. The obtained heat-treated product was subjected to centrifugation and the supernatant was collected. The precipitate was resuspended by adding an equal amount of water to the heat treatment solution, and subjected to centrifugation again to recover the supernatant. The RNA extract was obtained by combining the collected supernatants.

The obtained RNA extract was appropriately diluted and subjected to gel infiltration high performance liquid chromatography (hereinafter referred to as GPC-HPLC). _{TSKgel G3000PW XL} was used as the HPLC column, and a 7M urea / 50 mM Tris-HCl (pH 7.5) solution was used as the moving layer. A 260 nm ultraviolet light absorbing substance in the polymer region is detected. Schmidt Tannhäuser Schneider's method [J. Biol. Chem. The RNA solution quantified by 1946, 164, 747] (hereinafter referred to as the STS method) was subjected to the same analysis, and the amount of RNA in the extract was quantified from the area of the ultraviolet absorption peak.
By the above method, the RNA yields of the obtained mutant strains were calculated in the same manner as in Example 1, and strains having a higher RNA yield than the parent strain were selected.

Table 1 shows the results of selecting mutant strains showing high RNA yield using each of the above mutant strain acquisition methods.

In the KCl method, none of the 680 strains obtained had an RNA yield improved by 1.10 times or more of that of the parent strain.
In the rapamycin method, among the 560 strains obtained, there were 3 strains in which the RNA yield was improved to 1.10 times or more of that of the parent strain.

From the above results, it is clear that the method of the present invention can obtain a yeast mutant strain showing a high RNA yield with an efficiency of 10 times or more that of the KCl method and the rapamycin method, which are methods for obtaining a strain showing a known high RNA content. Became.

(Example 3) Examination of industrial RNA production using the mutant strain obtained by the present invention When industrial RNA production is performed, RNA yield is high on a small scale, but when scale-up is performed, the RNA yield is high. Strains with reduced RNA yields have been found and are often problematic.
Therefore, in this Example 3, in order to examine the feasibility of industrial RNA production using the mutant strain obtained in Example 1, the culture was scaled up and batch culture was performed using a fermenter. It was.

For examination, NV-9 (RNA yield in Example 1: 1.11 times the parent strain ratio) and NV-42 (RNA yield in Example 1: parent strain), which are RNA high-yield mutants obtained in Example 1, were examined. Three strains of NV-46 (RNA yield in Example 1: 1.12 times the parent strain ratio) were used. The above three strains were seed-cultured in a flask in advance, inoculated into a 3 L fermenter, and subjected to batch culture.
The medium composition is 4.2% glucose, 0.2% potassium chloride, 600ppm magnesium sulfate heptahydrate, 8.3ppm iron (III) chloride hexahydrate, 6.3ppm manganese chloride tetrahydrate, 0 .5 ppm copper sulphate / pentahydrate, 10 ppm zinc sulphate / heptahydrate, 2.2 g / L monoammonium phosphate, 5 g / L ammonium sulphate, 0.025% Adekanol LG-109. The culture conditions were medium liquid volume 1.5 L, liquid temperature 30 ° C., stirring rotation speed 1000 rpm, aeration 1.3 vvm, and oxygen gas was aerated depending on the situation so that the dissolved oxygen amount did not fall below 0.2 mg / L. did. In addition, 14% aqueous ammonia was added dropwise to maintain the pH at 3.2 or higher.
From the obtained culture broth, an RNA extract was obtained by the above-mentioned method, and the RNA yield was calculated.

As a result of this example, the ratio of the mutant RNA yield to the parent RNA yield in the extract obtained from each strain culture solution was as shown in Table 2.

All three strains obtained from the NBRC0988 strain according to the present invention were stable and showed a high RNA yield of 1.10 times or more as compared with the parent strain, which was equivalent to that of the small scale.
From the results of this example, it was clarified that the mutant strain selected by the present invention stably showed high RNA yield even in fermenter culture.

From the above, it was clarified that according to the present invention, a yeast mutant strain showing a higher RNA yield than the parent strain can be obtained with extremely high efficiency as compared with the existing mutant strain acquisition method.
In addition, the strain showing high RNA yield obtained by the present invention stably shows high RNA yield even when the scale of culture is increased, and thus it is clear that it is suitable for industrial RNA production. It was.

Claims (2)

A method for obtaining a Candida yeast mutant strain, which comprises the following steps (1) to (3) and has an improved RNA yield as compared with the parent strain.
(1) Step of mutagen treatment of yeast,
(2) A step of selecting a mutant strain having vanillin resistance from the yeast strains treated with mutagens.
(3) A step of obtaining a strain having a higher RNA yield than the parent strain from the selected strains. The step of obtaining a yeast mutant strain by the method according to claim 1.
Step of aerobically culturing the acquired yeast mutant strain,
The process of extracting RNA from cultured yeast,
A method for producing RNA, which comprises.