JPH11196859A - Mutant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable an RNA useful as a raw material of a medicine or a seasoning to be generated and accumulated in a fungal body at a high content by aerobically culturing Candida utilis mutant imparted with a highly low temperature sensitivity. SOLUTION: This mutant, e.g. Candida utilis 27B4317 (FREM P-16552), extremely reduced in the propagation rate at a low temperature and generating and accumulating an RNA in an amount of >=20 wt.% per the fungal body weight is obtained by subjecting Candida utilis CS 7529 (FREM P-3340), etc., to a mutation treatment. The mutant is aerobically cultured and the RNA is industrially obtained in an excellent productivity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a Candida utilis mutant which produces and accumulates 20% by weight or more of ribonucleic acid (hereinafter abbreviated as RNA) in cells, and a high RNA using the mutant. The present invention relates to a method for producing yeast containing yeast.

[0002]

2. Description of the Related Art RNA is used as a raw material for pharmaceuticals or umami seasonings such as 5'-inosinic acid and 5'-guanylic acid. For the production of this RNA, yeast cells cultured using molasses, liquid sugar, acetic acid or the like as a main carbon source are generally used. Yeasts have the advantage of high growth rate and high-density cultivation, and have been confirmed to be edible in Candida utilis and Saccharomyces cerevisiae. It is also possible to produce a yeast extract containing '-inosinic acid or 5'-guanylic acid, which is extremely suitable as a strain for producing RNA.

[0003] In recent years, the demand for yeast extract as a flavor seasoning has been expanding against the background of nature and health, and ribonucleic acid-rich yeast has been desired. However, the RNA content in known yeasts is as low as 5 to 12%,
Attempts to increase the content have been actively made up to the present. The attempt is as follows. 1. Investigation of culture conditions such as culture with the addition of the antibiotic anisomycin (Japanese Patent Publication No. 50-16875) and examination of the main carbon source (Japanese Patent Publication No. 57-29149). 2. Screening of yeast with high RNA content from nature (Japanese Patent Publication No. 55-49837); Improvement of strains by genetic modification represented by thiazine-based, acridine-based, oxazine-based dye resistance (Japanese Patent Publication No. 48-32350), KCl sensitivity (US Pat. No. 3,909,352), etc. Is done.

[0004]

However, even with the above-mentioned prior art, the bacterial strain obtained has an intracellular RNA content of only about 17% (dry cell weight of about 15 g / l). On the other hand, the present inventors have reported a mutant strain in which RNA content exceeds 14% by imparting low-temperature sensitivity to yeast cells (JP-B-56-46824, WO88 / 052).
No. 67), but a strain having a further increased RNA content and a good cell yield without changing the culture conditions has been desired.

[0005]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve such problems, and as a result, surprisingly, by imparting a high temperature sensitivity to yeast cells, the RNA content is surprisingly increased to 20% or more. It has been found that remarkably increased cells can be obtained and the cell yield is good, and the present invention has been achieved. That is, the present invention provides a mutant strain of Candida utilis that produces and accumulates RNA in an amount of 20% by weight or more per bacterial cell weight, and a method for producing a yeast with high RNA that aerobically cultures the mutant strain.

Hereinafter, the present invention will be described in detail. The mutant strain of the present invention is, for example, Candida utilis CS7529.
Mutant treatment using (FERMP-3340) or the like as a parent strain can be obtained by obtaining a low-temperature-sensitive mutant strain whose growth rate at a lower temperature is significantly lower than that of the parent strain. Specifically, using a mutagen such as ultraviolet light, ionizing radiation, nitrous acid, nitrosoguanidine, and ethyl methanesulfonate, there is no difference in the growth rate at room temperature on a synthetic medium, but the growth rate at low temperature Select strains that are significantly slower than the parent strain.
Such low-temperature-sensitive mutants are selectively concentrated by culturing at low temperature in a medium containing an antibiotic such as amphotericin B that acts only on yeast proliferating cells. From these strains, select those with lower temperature sensitivity and
By selecting those having a high content and a high cell yield, the desired mutant strain is obtained. Thus, in the present invention, Candida utilis 27B4317 (F
ERM P-16552) was obtained.

[0007] The mutant strain of the present invention, Candida utilis 27B4317, produces and accumulates a considerable amount of RNA of 20% by weight or more per cell weight, and is extremely suitable for the production of ribonucleic acid. Except for low temperature sensitivity, it is exactly the same as the general mycological properties of Candida utilis. The new mutant is F
Deposited as ERM P-16552. The following shows the mycological properties of the present yeast. 1. All edges, semi-lens-like ridges, smooth surface, dull gloss, creamy soft colonies form on YM agar. In a liquid medium containing glucose / yeast extract / peptone, turbidity and sedimentation were observed, and formation of a pellicle and a bacterial ring was not observed. 2. Form pseudohyphae on Dalmor plates. 3. No formation of ascospores was observed in each medium of sodium acetate agar slope, malt extract agar slope, V8 juice agar slope, and corn milk agar slope. 4. Ferment glucose, sucrose, raffinose, and do not ferment galactose, maltose, trehalose, lactose. 5. It assimilates glucose, sucrose, maltose, cellobiose, trehalose, raffinose, melezitose, xylose, ethanol, mannitol, lactic acid, citric acid and KNO3. 6. It grows well on a vitamin-free medium. 7. Oval or cylindrical cells (3.
5 to 4.5) × (7 to 13) μm. 8. It hardly grows at 20 ° C, but grows very slightly. 9. Grows well at 30-40 ° C.

[0008] The present invention further provides a method for producing a ribonucleic acid-rich yeast for aerobically culturing a mutant strain. In the medium for culturing the mutant strain of the present invention, as a carbon source, glucose, sucrose, acetic acid, ethanol, molasses, sulphite pulp waste liquor and the like are used, and as a nitrogen source, urea, ammonia, ammonium sulfate, ammonium chloride, Nitrates and the like are used. Ammonium phosphate, potassium chloride, magnesium sulfate, magnesium chloride and the like are used as the phosphoric acid, potassium and magnesium sources, and other inorganic salts such as zinc, copper, manganese and iron ions are added. In addition to the above, vitamins, amino acids, nucleic acid-related substances and the like need not be particularly added, but these may be added, or organic substances such as corn steep liquor, casein, yeast extract, meat extract, and peptone may be added. That is natural. The culture temperature is
21-38 ° C., preferably low temperature without significant growth delay, pH 3.5-8.0, especially 4.0-4.0.
6.0 is preferred. The culture format of the present invention may be either batch culture or continuous culture, but the latter is industrially employed.

[0009]

Next, the present invention will be described in detail with reference to examples.
The measurement of the RNA content was carried out according to the method of Schmidt Tannheiser Schneider [J. Biol. Chem. 1946 164 74
After extracting RNA by [7], the amount of RNA was determined and expressed as a percentage based on the dry weight of the cells.

Example 1 Candida utilis CS7529 (FERM P-
3340) was cultured in a test tube containing YPD medium (1% yeast extract, 2% polypeptone, 2% glucose) until the exponential growth phase. After collecting and washing the cells, Adelbe
Mutation treatment with nitrosoguanidine (NTG) was performed according to the method of Biochem. Biophys. Res.
18 788]. After washing the mutated cells and culturing them overnight in a YPD medium, the cells were treated with NTG. Then, NTG
Treated cells were harvested YPD medium so that 10 ⁷ ~ ⁸ cells / ml, was allowed to warm and cultured under 20 ° C. until the logarithmic growth phase. Amphotericin B was added thereto to a final concentration of 30 ppm, and the cells were further cultured at 20 ° C. for 3 hours.
The cells were collected, washed, diluted, and smear-cultured on a YPD agar medium. Next, this was used as a master plate, and a low-temperature sensitive strain was selected by a replica. The culture temperature at this time is 3
0 ° C and 20 ° C, the growth rate at 30 ° C is the same as that of the parent strain, but the growth at a limited temperature of 20 ° C is remarkably slow;
Alternatively, those that did not grow were obtained as cold-sensitive mutants. Candida utilis 27B43 is selected from these strains having a high RNA content and a good cell yield.
17 (FERMP-16552) was obtained.

Example 2 Candida utilis 27B4317 (FERM P
-16552) was preliminarily cultured in an Erlenmeyer flask containing a YPD medium, and this was inoculated in a 30-L fermenter by 1-2%. The composition of the medium is as follows: glucose 4%, monoammonium phosphate 0.3%, ammonium sulfate 0.161%, potassium chloride 0.137%, magnesium sulfate 0.08%, copper sulfate 1.6ppm, iron sulfate 14ppm, manganese sulfate 16p
pm and 14 ppm of zinc sulfate. Culture conditions are pH
4.0, culture temperature 24 ° C., aeration rate 1 vvm, stirring 400
The reaction was performed at rpm, and ammonia was added to control the pH. For comparison, the parent strain Candida utilis CS7529 (FERMP-3340) was similarly cultured. The results are shown in Table 1. As is clear from Table 1, the 27B4317 strain produced and accumulated a remarkable amount of RNA in the cells exceeding that of the CS7529 strain, had a high yield against saccharide cells, and the RNA content exceeded 20%.

[0012]

[Table 1]

[0013]

As described above, according to the present invention, a cold-sensitive Candida utilis mutant having an RNA content of 20% or more, and an RNA obtained by aerobically culturing the mutant can be obtained. A method for producing a yeast which accumulates in a large amount in a bacterial cell is provided, and industrial RNA productivity can be significantly improved.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:72)

Claims (4)

[Claims] 1. A Candida utilis mutant strain having low-temperature sensitivity and producing and accumulating ribonucleic acid in an amount of 20% by weight or more per cell weight. 2. The Candida utilis mutant strain is Candida utilis 27B4317 (FERM P-1).
6552). The Candida utilis mutant according to claim 1, wherein the strain is Candida utilis. 3. A method for producing a ribonucleic acid-rich yeast, which comprises aerobically culturing a Candida utilis mutant strain to produce and accumulate 20% by weight or more of ribonucleic acid in the yeast cells. . 4. The Candida utilis mutant strain is Candida utilis 27B4317 (FERM P-1).
The method for producing a ribonucleic acid-rich yeast according to claim 2, which is 6552).