KR101517558B1 - Saccharomyces cerevisiae with high RNA content and use thereof - Google Patents

Abstract

The present invention relates to a novel strain containing RNA at a high concentration, a natural flavor material containing the same, and a method for mass production of the strain.

Description

Highly concentrated RNA yeast strains and their use {Saccharomyces cerevisiae with high RNA content and use thereof}

The present invention relates to a novel strain containing RNA at a high concentration, a natural flavor material containing the same, and a method for mass production of the strain. The present invention also relates to a SPT15 mutant protein (Suppressor of Ty 15) derived from the strain and a gene encoding the same.

As the use of flavor materials widely used in the food industry has increased, a variety of materials have been developed. In recent years, there has been a decrease in preference for products made from chemical synthesis, so that monosodium glutamic acid (MSG), hydrolized vegetable protein Of natural flavor materials that can replace chemical ingredients and seasonings. Yeast extract is the natural flavor material.

Unlike other seasonings, in which only amino acids are the main tastes, yeast extract is rich in not only amino acids but also peptides, nucleic acids, vitamins and minerals. Therefore, it has excellent taste and rich taste, And can not be seen in other seasonings.

However, since yeast extract has an inherent yeast content, its usage is limited. Recently, there is a tendency to produce and use high-nucleoside yeast extract, which has stronger tastes and richness than conventional yeast extracts and is free from yeast odor. In order to produce a high-nucleate yeast extract, a large amount of precursor RNA that produces a 5'-nucleotide, which is a component of a nucleic acid seasoning, must be contained in the cells. The RNA content in yeast varies depending on the species, but generally has an RNA content of about 7 to 8%. When the nucleic acid in the yeast is extracted and chemically or enzymatically decomposed, nucleotides are produced. These nucleotides have qualities and thus become a raw material for a kind of nucleic acid seasoning. Therefore, in order to produce a yeast extract having many nucleotides, it is necessary to first select a yeast strain having a high RNA content or to improve the accumulation of RNA in the cells.

As a method for obtaining a cell having a high RNA content, Japanese Patent Application Laid-open No. 56-16824 discloses a technique for increasing the RNA content by culturing a low-temperature sensitive mutant using a continuous fermentation method. However, this method has a disadvantage in that it is difficult to produce a uniform product and the expense increases because mutation growth is relatively unstable. Methods for increasing the RNA content by controlling the culture environment, such as changing the growth medium composition or the medium additives, have also been studied. For example, Japanese Patent Application Laid-Open Nos. 40-15960, 50-16875, 45-15946 discloses a method in which an inorganic salt or an antibiotic substance is added to a medium to increase the RNA content. However, the method using such a medium additive has a problem in that the total RNA content finally obtained is decreased because the cell growth is reduced. In addition, Korean Patent No. 10-0442574 discloses a yeast mutant having high RNA content after treating with a chemical mutagen to induce mutation. However, the strain thus selected has a higher RNA content per dry cell than that of the parent strain Although it was high, its effect was not remarkable. This is because raising the RNA content in yeast strains is very difficult to obtain compared to increasing the yield of other metabolites.

Meanwhile, gTME (Global Transcriptome Machinery Engineering) was started to increase the efficiency of biofuel production by using yeast, and it was found that by reprogramming profiles by converting major proteins that regulate transcripts to generate various phenotypes (Alper et al., SCIENCE, 314: 1565-1568, 2006). The production of metabolites in yeast is not determined by a single gene, but dozens to hundreds of proteins that regulate gene expression constitute a massive chemical reaction network that interacts with sophisticated control schemes. gTME is a new technique for replacing cells in general, with about 300 genes being significantly transformed, mutation using gTME technique has increased productivity by 70%, production is increased by 15%, and ethanol fermentation ability is superior (Alper et al., SCIENCE, 314: 1565-1568, 2006). However, no studies have been reported so far on the development of high-nucleic acid yeast mutants with increased RNA content by applying gTME.

The present inventors selected high-concentration RNA yeast mutants having increased RNA content by enhancing the biosynthesis of RNA precursors using gTME technology, analyzed the RNA content and growth characteristics thereof, and confirmed that they are applicable to industry, thus completing the present invention.

It is an object of the present invention to provide a yeast strain having a high RNA content in cells. The strain may be used as a natural flavor material which has no odor and is excellent in taste and richness.

Accordingly, another object of the present invention is to provide a natural seasoning composition comprising the extract of the yeast strain.

It is still another object of the present invention to provide a method for mass production of the yeast strain.

Yet another object of the present invention is to provide an SPT15 mutant protein derived from the yeast strain.

It is another object of the present invention to provide a gene encoding the SPT15 mutant protein.

Yet another object of the present invention is to provide a vector comprising the gene encoding the SPT15 mutant protein.

It is still another object of the present invention to provide a method for producing a high-RNA-content yeast strain, which comprises transforming a vector containing the gene into a yeast strain.

The present invention relates to a novel strain containing RNA at a high concentration and its use, and the strain can be used as a natural flavor material having no odor and excellent in taste and richness.

In one embodiment, the present invention relates to a high-nucleic acid yeast strain with increased RNA content, comprising a mutated SPT15 (Suppressor of Ty 15).

The high-nucleic acid yeast strain in which the RNA content is increased in the present specification is at least 10% by weight, preferably at least 20% by weight, more preferably at least 20% by weight, By weight, more preferably at least 25% by weight. According to a preferred embodiment of the present invention, the yeast mutant of the present invention contains 20 to 30% by weight of RNA per dried cell.

Cells inherently regulate 1,000 genes at a time, with yeast having over 75 proteins that control gene expression (Fig. 1). Among them, TATA box binding protein, SPT15, functions to regulate the activities of RNA polymerase I, II, and III transcription factors. The high-nucleotide yeast strain of the present invention includes the mutated SPT15, thereby corresponding to a yeast mutant strain in which the RNA content is increased by enhancing the biosynthesis of the RNA precursor. The amino acid sequence of SPT15 is shown in SEQ ID NO: 1, and the yeast mutant of the present invention includes a mutation produced by substitution, deletion, addition, duplication, or inversion of at least one of the amino acids listed in SEQ ID NO:

The present inventors constructed a mutated SPT15 gene using PCR-mediated random mutagenesis and transformed it into yeast to isolate yeast strains containing high concentration of RNA. They were grown under various culture conditions . In a specific example of the present invention, a random mutation of SPT15, a TATA binding protein of yeast, was induced to generate about 50,000 Ura + mutants. To the medium containing normal medium and KCl, tooth picking was performed, 1,114 strains that did not grow well were selected in the medium. RNA was quantitated by orcinol method and strains # 132-484, which showed the highest RNA content, were selected. The RNA content of the strain 132 ~ 484 was 36.6 hours and the RNA content was increased by 27.6% compared to the parent strains in the culture conditions using peptone as a carbon source and glucose as a carbon source. It was also confirmed that the RNA content was further increased by 16.8% under the condition of pH 4.5. When the culture was performed using the 1 L fermenter under the above conditions, the RNA content was increased by about 23.6%. Sequencing of SPT15 confirmed that the 13th, 16th and 126th amino acids were replaced with alanine to glutamic acid, isoleucine to lysine, and alanine to valine. Furthermore, it has shown that yeast extract, which is a natural natural ingredient, can be used more economically.

Thus, according to a preferred embodiment of the invention, the strain of the invention comprises an amino acid sequence mutated within the wild-type SPT15 amino acid sequence. The mutated SPT15 gene can be introduced into a yeast cell in the form of a plasmid or introduced into the genomic DNA of a yeast cell. Preferably, the yeast strain is Saccharomyces spp., More preferably Saccharomyces cervisiae. More preferably a strain containing SPT15 mutated with Y2805 as a parent strain in Saccharomyces cerevisiae.

In one embodiment, the strain of the present invention may be a high-nucleic acid yeast strain having an increased RNA content, comprising the SPT15 mutant protein consisting of the amino acid sequence of SEQ ID NO: 5 or a gene encoding the SPT15 mutant protein. In the SPT15 mutant protein of SEQ ID NO: 5, alanine (Ala, A) at the thirteenth position is substituted with glutamic acid (Glu, E) in the wild type SPT15 of the parent strain (SEQ ID NO: (Ile, I) is substituted with lysine (Lys, K) and alanine (Ala, A) at 126th position is substituted with valine (Val, V). Preferably, the nucleotide sequence of the gene encoding the SPT15 mutant protein of SEQ ID NO: 5 is represented by SEQ ID NO: 6.

Preferably, the strain of the present invention is a carbon source and is capable of growing in the presence of glucose, galactose, molasses or sucrose, and may contain peptone, ammonium chloride (NH ₄ Cl ⁺ ), ammonium phosphate _{(NH 4 H 2 PO 4)} , glycine, can be grown in the presence of glutamic acid or ammonium sulfate. It is also possible to grow at a pH of 4.5 to 6.5. However, the scope of the present invention is not limited to the above culture conditions, and culture conditions can be changed according to the choice of a person skilled in the art.

In another aspect, the present invention relates to a SPT15 mutant protein consisting of the amino acid sequence of SEQ ID NO: 5.

The present invention also relates to a gene encoding the SPT15 mutant protein. For example, the gene may comprise the nucleotide sequence of SEQ ID NO: 6.

The SPT15 mutant protein provided by the present invention is derived from the high-nucleic acid yeast strain # 132-484 in which the RNA content selected in the present invention is increased. In the SPT15 mutant protein of SEQ ID NO: 5, alanine (Ala, A) at the thirteenth position is substituted with glutamic acid (Glu, E) in the wild type SPT15 of the parent strain (SEQ ID NO: (Ile, I) is substituted with lysine (Lys, K) and alanine (Ala, A) at 126th position is substituted with valine (Val, V).

Preferably, the nucleotide sequence of the gene encoding the SPT15 mutant protein of SEQ ID NO: 5 is represented by SEQ ID NO: 6. In the gene of SEQ ID NO: 6, the 38th C is replaced with A, the 47th T is replaced with A, and the 377th C is replaced with T in the gene sequence (SEQ ID NO: 2) of wild type SPT15 of the parent strain will be.

In another aspect, the present invention relates to a vector comprising a gene encoding the mutant protein.

In the present invention, various vectors such as plasmids, viruses, and cosmids can be used as the vectors. The recombinant vector includes a cloning vector and an expression vector. A cloning vector is a replicon that includes a replication origin, for example, a replication origin of a plasmid, phage or cosmid, and in which the fragment to which another DNA fragment is attached can be cloned. Expression vectors have been developed for use in synthesizing proteins, and conventional ones that are used in the art to express foreign proteins in plants, animals, or microorganisms can be used. The recombinant vector may be constructed by a variety of methods known in the art.

The recombinant vector is usually a carrier into which a fragment of foreign DNA is inserted, and usually means a fragment of double-stranded DNA. The recombinant vector may be operably linked to a transcriptional and detoxification regulatory sequence that functions in a selected expression host to increase the expression level of the transgene in the host cell. The recombinant vector is a gene construct comprising an essential regulatory element operably linked to the expression of a gene insert in a cell of an individual. Standard recombinant DNA techniques may be used to produce such gene constructs.

The recombinant vector is not particularly limited as long as it has a function of producing a desired protein. However, a vector capable of producing a foreign protein having a form similar to a natural state while having a strong expression ability and a promoter showing strong activity is preferable. The recombinant vector preferably contains at least a promoter, an initiation codon, a gene encoding a desired protein, a termination codon, and a terminator. In addition, DNA encoding the signal peptide, an enhancer sequence, a non-toxin region at the 5'-terminal and 3'-terminal of the desired gene, a selective marker region, or a replicable unit may suitably be contained.

In the present invention, it is preferable that the host cell is yeast, but the carrier DNA can be amplified by introduction from E. coli or the like before transformation into yeast.

In another aspect, the present invention relates to a method for producing a high-RNA-content yeast strain with high RNA content, which comprises transforming a vector containing the gene into a yeast strain.

As a method for preparing a transformant by introducing a recombinant vector into a host cell, an introduction method well known in the art can be used. When the host cell is a prokaryotic cell, a CaCl ₂ method or an electroporation method can be used , Transfection, calcium phosphate precipitation, electroporation, liposome-mediated transfection (e. G., Lipofection), microinjection, and particle bomoardment, YAC in the case where the host cell is a eukaryotic cell Yeast spheroid / cell fusion used, and the like, but the present invention is not limited thereto.

In another aspect, the present invention relates to a yeast strain transformed with said vector.

In another embodiment, the present invention relates to a natural seasoning composition comprising an extract of said yeast strain.

Preferably, the yeast strain extract may be one obtained by decomposing the yeast strain of the present invention chemically or enzymatically. For example, the yeast cells can be extracted by a method of treating the yeast cells with a cell wall degrading enzyme, a nucleic acid degrading enzyme, an AMP converting enzyme, or a proteolytic enzyme, followed by enzymatic degradation, or by disrupting the cells using ultrasound or the like . Such a yeast degradation process can be carried out at a pH of 4 to 8 at a temperature of 50 to 70 ° C for 45 to 60 hours, but the specific decomposition conditions can be changed according to the selection of a person skilled in the art. Once the yeast digestion reaction is complete, the process of removing specific yeast odor through the treatment of activated charcoal and keeping the chromaticity bright may be additionally carried out.

Also, the present invention provides a natural seasoning composition comprising yeast culture, extract or extract prepared by the above-mentioned method. The composition may be processed into various nutritional supplements, health supplements, and the like, and may contain other ingredients, etc., which can give a synergistic effect to the main effect within a range not impairing the intended main effect of the present invention. For example, additives such as perfume, coloring agent, bactericide, antioxidant, preservative, moisturizing agent, thickening agent, inorganic salt, emulsifier and synthetic polymer substance may be further added for improvement of physical properties. In addition, it may further contain auxiliary components such as water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymeric polysaccharides and seaweed extract. The above components may be mixed and selected without difficulty by those skilled in the art depending on the purpose of formulation or use, and the amount thereof may be selected within a range that does not impair the objects and effects of the present invention.

In another aspect, the present invention relates to a method for mass production of the yeast strain.

Preferably, the yeast strains glucose, galactose, molasses and the number of carbon atoms selected from the group consisting of sucrose, and peptone, NH ₄ Cl ^+, NH ₄ H ₂ PO _4, glycine, glutamic from the group consisting of acid and ammonium sulfate In a medium containing a selected nitrogen source. The present invention also relates to a method for mass production of high-yield yeast strains.

The yeast strain of the present invention is a strain having an RNA content of 20% by weight or more, preferably 25% by weight or more, as compared with the parent strain, and it is said that the effect is very excellent considering that it is very difficult to increase the intracellular RNA content . The thus-obtained high-nucleate yeast is safe as well as excellent in quality as a natural flavor material, and thus can be usefully used as a raw material for natural seasoning.

By developing the Jungmi material, which has relied on foreign countries using the high-concentration RNA yeast of the present invention, it is possible to lower the dependency of the product on foreign countries, to supply the natural flavor material made of the high-concentration RNA yeast at an inexpensive price and further to find new functional materials Do.

1 is a view showing SPT15 (TATA box binding protein; TBP).
Figure 2 (A) shows the plasmid plasmid pRS316-GCYH2gR and (B) shows the RS316-spt15 mutant library.
Figure 3 shows the ura + mutant strain grown on YSCD-ura medium.
Fig. 4 shows tooth picking results on YPD medium (left) and KCl medium (right).
5 shows the parent strain (Y2805) and the mutant strains (132-484) grown on the YPD plate (A) and the KCl-containing plate (B).
Fig. 6 shows the cell concentration (A), RNA concentration (B) and RNA content (C) according to the carbon source of the parent strain and the mutant strain. DCW (dry cell weight) represents the dry cell weight.
7 shows the cell concentration (A), RNA concentration (B) and RNA content (C) according to the nitrogen source of parent strain and mutant strain. DCW (dry cell weight) represents the dry cell weight.
Figure 8 shows the cell concentration (A), RNA concentration (B) and RNA content (C) according to the pH of the mutant strains (# 132-484). DCW (dry cell weight) represents the dry cell weight.
Figure 9 shows the growth curves (?,?), RNA concentration (?,?) And RNA content (?,?) Of the mutant strains (# 132-484) and parent strain (S. cerevisiae Y2805) . □, ○, and ◇ indicate the results in this parent strain, and ■, ●, and ◆ indicate the results in this mutant strain.
10 shows the SPT15 protein structure and mutation positions of (A) parent strain (S. cerevisiae Y2805) and (B) mutant strains (# 132-484).

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  One. SPT15  Building mutation libraries

RT-PCR was used to generate the SPT15 mutant. First strand cDNA was synthesized by transcribing 2 μg total RNA with andom hexamer and M-MuLV reverse transcriptase (Promega, Madison, WI). PCR was performed at 95 ° C for 1 minute, at 55-60 ° C for 1 minute, and at 72 ° C (20 cycles depending on the length of DNA, RT-PCR and 30 cycles of regular PCR). The whole ORF (open reading frame) of wild type SPT15 (SPT15wt) was PCR-amplified using sense primer (5'-gtagggatcctgagatggccgatgaggaacgtt-3 ') and antisense primer (5'-gtaggaattctcacatttttctaaattcacttag-3') using genomic DNA as template, -T easy vector to prepare pT-SPT15. The SPT15 mutant library was generated using the GeneMorph II random mutagenesis kit (Stratagene, La Jolla, Calif.) (Template: pT-SPT15, primer: same as above). The mutant gene cleaved with restriction enzymes BamHI and EcoRI was inserted into the plasmid pRS316-GCYH2gR (Fig. 2A) and placed under the control of glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3P) and galactose-1-phosphate uridyl transferase terminator (GAL7T). The plasmid thus prepared (Figure 2B) was transformed into E. coli DH5α and incubated at 30 ° C to prevent outgrowth of fast-growing cells. The library plasmid (500 μg) was transformed into S. cerevisiae Y2805 [Matα, pep4: : his3, pRB- [Delta] 1.6R, Can1, his3-20, ura3-52] . ≪ / RTI >

Example  2. Yeast Transformation

All plasmids used for yeast transformation were prepared without RNA digestion and a mixture of DNA and RNA was used for yeast transformation. S. cerevisiae Y2805 was incubated overnight, and 20 ml of YPD was mixed with 200 ml of YPD, incubated at 30 ° C for 2 hours, and the YPD and tubes were stored in four 50 ml tubes for 5 min at room temperature (a). The pellet was collected into one 50 ml tube, washed with 1 X TE, and then the pellet was mixed again with 4 ml 1 x SiAc / TE. After incubation at room temperature for 10 minutes, the cell suspension was transferred to a 100 ml Erlenmeyer flask, and 500 μg of SPT15 mutant library DNA was added. 28 ml of PEG / LiAc solution (22.6 ml of 50%, 2.8 ml of 10 X SiAc, 2.8 ml of 10 X TE; 40% PEG, 1 X LiAc, 1 X TE, final) was added and incubated at room temperature for 30 minutes. And then occasionally shaken at 42 ° C for 10 minutes, followed by quenching at room temperature in a water bath. The cell suspension was transferred to a 50 ml tube, and the cell was submerged at 3000 rpm for 5 minutes. Then, the cell was washed with 50 ml 1 × TE (b), and the cells were again mixed with 200 ml of YPD stored in step (a). The number of cells was measured (c). Cells were cultured at 30 ° C for 6 hours, and the number of cells was counted. Spinning was carried out in four 50 ml tubes at room temperature for 5 minutes. Add the washing cell to the appropriate volume of 1 X TE with 50 ml of 1 X TE, and mix 100 ml of glycerol in 1/3 (5 ml) volume (total volume: 15 ml, depending on the number of cells in step (b) And cells were collected and stored at -80 ° C.

Example  3. Screening mutant libraries

YSCD-ura medium (YSCD medium, 0.67% yeast nitrogen base without amino acid, amino acid supplement mixture and 2% dextrose, and addition of 1.5% bacto-agar for solid plate, with metabolic gene ura3 as a selective maker; MP, OH) were cultured. 1 ml of the mutant library was removed from the deep freezer and suspended in 7 ml of 1 × TE buffer. 400 μl aliquots were suspended in YSCD-ura plates and incubated at 30 ° C for 3 days. After 3 days, transformed colonies grew .

The mutant strain, which does not grow well in alkaline RNase inhibitor medium, was expected to inhibit acidic RNase activity or to accumulate high-concentration RNA at acidic pH, (1% bacto yeast extract, 2% bactopeptone and 2 w / v% glucose, and 1% bacteriophage for solid plate) in the presence of 1.5% KCl in the medium to inhibit the alkaline RNase activity, Additionally containing agar, Difco, MI) And the strains that did not grow well in the KCl medium were visually selected.

Example  4. RNA  dose

10 ml of the bacterial culture was centrifuged at 10000 RPM for 5 minutes to remove the supernatant. The precipitate was suspended in 10% PCA and heated at 90 占 폚 for one hour. 650 μl of orcinol solution (Sigma-Aldrich, 0.5% Fe 2 Cl 3) and 750 μl of 35% hydrochloric acid were added to 100 μl of the test solution, and the mixture was heated at 95 ° C. for 20 minutes, and spectrophotomete (biomate 3, thermo, USA) and compared with the standard curve of the RNA sample (Roche).

Example  5. Fermentation conditions

In order to test the RNA accumulation ability according to the culture conditions of the selected high concentration RNA strain strain # 132-484, the RNA content was measured under different conditions of the carbon source and the nitrogen source pH of the culture, and 1 L fermenter (LiFlusGX, biotron, Korea) And the RNA content was measured. All cultures were carried out at 30 ° C and 200 RPM for 36 hours.

Experiment result

1. Selection of strains

3.2 x 10 ⁶ mutant library of S. cerevisae was generated using gTME (Fig. 3), of which 5 x 10 ⁴ transformed ura + mutant strains grown on YSCD-ura medium (Fig. 4). Among them, about 1,114 strains were not grown in the KCl-containing medium. The strains were quantitated by the orcinol method (# 5). Finally, strains # 132-484 having high RNA content were selected (FIG. The sequence of SPT15 protein from strain # 132-484 was analyzed by Macrogen (Seoul, Korea), and mutation was confirmed in A13E, I16K and A126V (FIG. 10; SEQ ID NO: 5 and SEQ ID NO: 6).

2. Analysis of culture conditions

Growth curves and RNA contents were measured after 36 hours of changing the carbon source (glucose, galactose, molasses, sucrose, lactose). As a result, # 132-484 strain showed the fastest growth in galactose and sucrose medium, I did. The RNA concentration (mg-RNA / ml-culture) was similar in all but the lactose medium, but was the highest at about 2.08 mg / ml in the glucose medium. RNA content (mg-RNA / mg-DCW × 100) was high in glucose and molasses medium and was the highest in glucose medium (26.7%) (FIG.

Growth curve and RNA content were measured after culturing for 36 hours by changing the nitrogen source (peptone, NH ₄ Cl ⁺ NH ₄ H ₂ PO ₄ , glycine, glutamic acid, ammonium sulfide) It was also the best. RNA content was lowest in peptone medium, but the RNA concentration of peptone medium was the best because RNA concentration of other media was much lower than peptone medium (FIG. 7).

 The growth curve and the RNA content were measured after changing the pH (4.5 to 6.5) for 36 hours, and the RNA concentration was the highest at pH 4.5 (about 2.43 mg / ml) (FIG. 8).

 The carbon source was glucose and the nitrogen source was fermented with peptone at pH 4.5. During the incubation period, the cell concentration was 20 ~ 30% lower than the parent strain, but the RNA concentration was similar. Therefore, the RNA content of the parent strain was 20.8% and the RNA of the # 132-484 strain was about 25.7% at 36 hours of culture. The mutant strain of the present invention increased the RNA content by about 23.6% And the content was maintained at about 20% (Fig. 9).

<110> Ewha University Industry Collaboration Foundation <120> Saccharomyces cerevisiae with high RNA content and use thereof <130> DPP20135111KR <150> KR10-2012-0111714 <151> 2012-10-09 <160> 6 <170> Kopatentin 1.71 <210> 1 <211> 240 <212> PRT <213> Saccharomyces cerevisiae <220> <221> PEPTIDE &Lt; 222 > (1) .. (240) <223> amino acid sequence of SPT15 <400> 1 Met Ala Asp Glu Glu Arg Leu Lys Glu Phe Lys Glu Ala Asn Lys Ile   1 5 10 15 Val Phe Asp Pro Asn Thr Arg Gln Val Trp Glu Asn Gln Asn Arg Asp              20 25 30 Gly Thr Lys Pro Ala Thr Thr Phe Gln Ser Glu Glu Asp Ile Lys Arg          35 40 45 Ala Ala Pro Glu Ser Glu Lys Asp Thr Ser Ala Thr Ser Gly Ile Val      50 55 60 Pro Thr Leu Gln Asn Ile Val Ala Thr Val Thr Leu Gly Cys Arg Leu  65 70 75 80 Asp Leu Lys Thr Val Ala Leu His Ala Arg Asn Ala Glu Tyr Asn Pro                  85 90 95 Lys Arg Phe Ala Ala Val Ile Met Arg Ile Arg Glu Pro Lys Thr Thr             100 105 110 Ala Leu Ile Phe Ala Ser Gly Lys Met Val Val Thr Gly Ala Lys Ser         115 120 125 Glu Asp Asp Ser Lys Leu Ala Ser Arg Lys Tyr Ala Arg Ile Ile Gln     130 135 140 Lys Ile Gly Phe Ala Ala Lys Phe Thr Asp Phe Lys Ile Gln Asn Ile 145 150 155 160 Val Gly Ser Cys Asp Val Lys Phe Pro Ile Arg Leu Glu Gly Leu Ala                 165 170 175 Phe Ser His Gly Thr Phe Ser Ser Tyr Glu Pro Glu Leu Phe Pro Gly             180 185 190 Leu Ile Tyr Arg Met Val Lys Pro Lys Ile Val Leu Leu Ile Phe Val         195 200 205 Ser Gly Lys Ile Val Leu Thr Gly Ala Lys Gln Arg Glu Glu Ile Tyr     210 215 220 Gln Ala Phe Glu Ala Ile Tyr Pro Val Leu Ser Glu Phe Arg Lys Met 225 230 235 240 <210> 2 <211> 723 <212> DNA <213> Saccharomyces cerevisiae <220> <221> gene <222> (1). (723) <223> nucleic acid sequence encoding SPT15 <400> 2 atggccgatg aggaacgttt aaaggagttt aaagaggcaa acaagatagt gtttgatcca 60 aataccagac aagtatggga aaaccagaat cgagatggta caaaaccagc aactactttc 120 cagagtgaag aggacataaa aagagctgcc ccagaatctg aaaaagacac ctccgccaca 180 tcaggtattg ttccaacact acaaaacatt gtggcaactg tgactttggg gtgcaggtta 240 gatctgaaaa cagttgcgct acatgcccgt aatgcagaat ataaccccaa gcgttttgct 300 gctgtcatca tgcgtattag agagccaaaa actacagctt taatttttgc ctcagggaaa 360 atggttgtta ccggtgcaaa aagtgaggat gactcaaagc tggccagtag aaaatatgca 420 agaattatcc aaaaaatcgg gtttgctgct aaattcacag acttcaaaat acaaaatatt 480 gtcggttcgt gtgacgttaa attccctata cgtctagaag ggttagcatt cagtcatggt 540 actttctcct cctatgagcc agaattgttt cctggtttga tctatagaat ggtgaagccg 600 aaaattgtgt tgttaatttt tgtttcagga aagattgttc ttactggtgc aaagcaaagg 660 gaagaaattt accaagcttt tgaagctata taccctgtgc taagtgaatt tagaaaaatg 720 tga 723 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> sense primer <400> 3 gtagggatcc tgagatggcc gatgaggaac gtt 33 <210> 4 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> antisense primer <400> 4 gtaggaattc tcacattttt ctaaattcac ttag 34 <210> 5 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of SPT15 mutant <400> 5 Met Ala Asp Glu Glu Arg Leu Lys Glu Phe Lys Glu Glu Asn Lys Lys   1 5 10 15 Val Phe Asp Pro Asn Thr Arg Gln Val Trp Glu Asn Gln Asn Arg Asp              20 25 30 Gly Thr Lys Pro Ala Thr Thr Phe Gln Ser Glu Glu Asp Ile Lys Arg          35 40 45 Ala Ala Pro Glu Ser Glu Lys Asp Thr Ser Ala Thr Ser Gly Ile Val      50 55 60 Pro Thr Leu Gln Asn Ile Val Ala Thr Val Thr Leu Gly Cys Arg Leu  65 70 75 80 Asp Leu Lys Thr Val Ala Leu His Ala Arg Asn Ala Glu Tyr Asn Pro                  85 90 95 Lys Arg Phe Ala Ala Val Ile Met Arg Ile Arg Glu Pro Lys Thr Thr             100 105 110 Ala Leu Ile Phe Ala Ser Gly Lys Met Val Val Thr Gly Val Lys Ser         115 120 125 Glu Asp Asp Ser Lys Leu Ala Ser Arg Lys Tyr Ala Arg Ile Ile Gln     130 135 140 Lys Ile Gly Phe Ala Ala Lys Phe Thr Asp Phe Lys Ile Gln Asn Ile 145 150 155 160 Val Gly Ser Cys Asp Val Lys Phe Pro Ile Arg Leu Glu Gly Leu Ala                 165 170 175 Phe Ser His Gly Thr Phe Ser Ser Tyr Glu Pro Glu Leu Phe Pro Gly             180 185 190 Leu Ile Tyr Arg Met Val Lys Pro Lys Ile Val Leu Leu Ile Phe Val         195 200 205 Ser Gly Lys Ile Val Leu Thr Gly Ala Lys Gln Arg Glu Glu Ile Tyr     210 215 220 Gln Ala Phe Glu Ala Ile Tyr Pro Val Leu Ser Glu Phe Arg Lys Met 225 230 235 240 <210> 6 <211> 723 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > nucleic acid sequence encoding SPT15 mutant <400> 6 atggccgatg aggaacgttt aaaggagttt aaagaggaaa acaagaaagt gtttgatcca 60 aataccagac aagtatggga aaaccagaat cgagatggta caaaaccagc aactactttc 120 cagagtgaag aggacataaa aagagctgcc ccagaatctg aaaaagacac ctccgccaca 180 tcaggtattg ttccaacact acaaaacatt gtggcaactg tgactttggg gtgcaggtta 240 gatctgaaaa cagttgcgct acatgcccgt aatgcagaat ataaccccaa gcgttttgct 300 gctgtcatca tgcgtattag agagccaaaa actacagctt taatttttgc ctcagggaaa 360 atggttgtta ccggtgtaaa aagtgaggat gactcaaagc tggccagtag aaaatatgca 420 agaattatcc aaaaaatcgg gtttgctgct aaattcacag acttcaaaat acaaaatatt 480 gtcggttcgt gtgacgttaa attccctata cgtctagaag ggttagcatt cagtcatggt 540 actttctcct cctatgagcc agaattgttt cctggtttga tctatagaat ggtgaagccg 600 aaaattgtgt tgttaatttt tgtttcagga aagattgttc ttactggtgc aaagcaaagg 660 gaagaaattt accaagcttt tgaagctata taccctgtgc taagtgaatt tagaaaaatg 720 tga 723

Claims (13)

(Suppressor of Ty 15) mutant protein consisting of the amino acid sequence of SEQ ID NO: 5 and exhibiting an RNA content increasing activity in yeast.
A gene encoding the SPT15 mutant protein of claim 1.
3. The gene according to claim 2, wherein the gene comprises the nucleotide sequence of SEQ ID NO: 6.
A vector comprising the gene of claim 2.
A method for producing a high-RNA-content yeast strain having an increased RNA content, which comprises transforming a vector comprising the gene of claim 2 into a yeast strain.
A high-nucleic acid yeast strain having increased RNA content, comprising an SPT15 mutant protein consisting of the amino acid sequence of SEQ ID NO: 5 or a gene encoding the SPT15 mutant protein.
The strain according to claim 6, capable of growing in the presence of glucose as a carbon source in the presence of galactose, molasses or sucrose.
7. The strain according to claim 6, capable of growing in the presence of peptone as a nitrogen source.
7. The strain according to claim 6, capable of growing at a pH of 4.5 to 6.5.
The strain according to claim 6, wherein the RNA content is increased by at least 20% by weight relative to the parent strain.
The strain according to claim 6, which contains 20 to 30% by weight of RNA per dried cell.
12. A natural seasoning composition comprising an extract of the yeast strain according to any one of claims 6 to 11.
The yeast strain according to any one of claims 6 to 11, which is selected from the group consisting of a carbon source selected from the group consisting of glucose, galactose, molasses and sucrose, and a group consisting of peptone, ammonium chloride, ammonium phosphate, glycine, glutamic acid and ammonium sulfate In a culture medium containing a nitrogen source selected from the group consisting of: &lt; RTI ID = 0.0 &gt; a &lt; / RTI &gt;

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