High-throughput rapid breeding method for high-ribonucleic acid yeast

Abstract

This is a high-throughput rapid breeding method for high-ribonucleic acid yeast. The provided high-throughput breeding method for high-ribonucleic acid yeast includes the steps of: (1) constructing a microbial mutant library; (2) restoring the cells obtained in step (1); (3) adaptively evolving the mutant library; and (4) obtaining high-RNA yeast by high-throughput screening of the evolutionary library. By employing the provided method, high-RNA yeast strains can be efficiently screened and dominant strains can be rapidly bred, and the RNA content of the screened dominant strains can reach 15% or more.

Description

Technical Field

[0001] The present invention relates to biotechnology, and specifically to a high-throughput screening method for microbial strains.

Background Art

[0002] Ribonucleic acid (i.e., RNA) is one of the important biopolymers and is widely applied in fields such as food seasonings, nutrition and health care, and biomedicine. Currently, the industrial production of RNA is mainly carried out by extracting from pure-cultured Candida yeast, Saccharomyces cerevisiae or brewer's yeast. Saccharomyces cerevisiae is a food safety level (GRAS) microorganism that does not produce any toxins, and has a fast growth and subculture rate, is easy to culture and rich in nutritional value, so it is the most ideal RNA source recognized.

[0003] Since the RNA content of Saccharomyces cerevisiae is relatively low, averaging 6% - 8%, the costs of products such as RNA and nucleotides derived from Saccharomyces cerevisiae are high. In order to reduce production costs and enhance the competitiveness of products, it is necessary to further improve the RNA content of yeast strains.

Summary of the Invention

Problems to be Solved by the Invention

[0004] In the existing technology, traditional screening methods are adopted, which makes it difficult to achieve large-scale and efficient screening, and moreover, the operation is complicated. In response to the problems existing in the existing technology, the present invention provides an efficient and rapid breeding method for high-nucleic acid yeast.

Means for Solving the Problems

[0005] The present invention provides a high-throughput breeding method for high ribonucleic acid yeast, and the method includes: Step (1) of constructing a microbial mutant library of yeast mutant cells, Step (2) involves restoring the bacterial cells obtained in step (1), Step (3) of adaptively evolving the mutant library, The procedure includes (4) obtaining high RNA yeast by high-throughput screening of an adaptive evolution library.

[0006] Preferably, in step (1), the step of constructing a microbial mutant library employs chemical mutagenesis, physical mutagenesis, or a combination of chemical mutagenesis and physical mutagenesis.

[0007] More preferably, the chemical mutagenesis agent is one or more of ethyl methanesulfonate, diethyl sulfate, and sodium azide. More preferably, the effective concentration of the chemical mutagenesis agent is 1-6% (w / v). The percentage of the effective concentration of the chemical mutagenesis agent is expressed as w / v, which specifically means that the mass of the chemical mutagenesis agent contained in 100 mL of the chemical mutagenesis agent solution is 1-6 g. Here, preferably, the volume ratio of the chemical mutagenesis agent to the yeast solution is 0.5-2.5:1, more preferably 1:1.

[0008] More preferably, the duration of action is 0.5 to 2 hours.

[0009] More preferably, physical mutagenesis employs one or more combinations of ultraviolet radiation and ambient pressure room temperature plasma. More preferably, the time of physical mutagenesis is 30s to 90s. More preferably, the output power of the ambient pressure room temperature plasma is 80-120 W and the gas flow rate is 8-15 L / min, preferably the output power of the ambient pressure room temperature plasma is 100 W and the gas flow rate is 10 L / min. More preferably, the output of ultraviolet radiation is 15W to 20W, and the irradiation distance is 20 to 30cm.

[0010] Preferably, in step (2), the bacterial cells are cultured statically in a culture medium, More preferably, the culture medium is YPD medium. More preferably, the pH of the culture medium is 5.0 to 6.0. More preferably, the culture temperature is 28-32°C. More preferably, the incubation time is 30 to 60 minutes.

[0011] More preferably, in step (3), the restored bacterial cells obtained in step (2) are cultured in an evolutionary medium containing an inhibitory substance, and the culture solution is collected and subcultured in a new evolutionary medium, and this subculture is repeated 50 to 150 times. Preferably, the inhibitory substance is one or more of the following: 6-azauracil, 8-azaguanine, cycloheximide, 6-mercaptopurine, diaminopurine, 5-bromouracil, 5-fluorouracil. More preferably, the concentration of inhibitory substances in the culture medium after subculturing increases. More preferably, during the subculturing process, the concentration of the inhibitory substance in the culture medium increases by 50 to 1000 mg / L. More preferably, the culture is performed for 20-30 hours before subculturing, preferably for 24 hours. More preferably, the culture temperature is 28-33°C, and the culture is performed with shaking at 150-220 rpm. More preferably, the concentration of the inhibitory substance is 50 mg to 1000 mg / L, and more preferably, the concentration of the inhibitory substance is 50 mg to 500 mg / L. More preferably, the initial concentration of the inhibitory substance is 50 mg to 100 mg / L.

[0012] More preferably, in step (4), the mutagenic library is stained with an RNA fluorescent dye, and strains with high fluorescence intensity are selected to obtain high RNA yeast. More preferably, the RNA fluorescent dye is one or more combinations of SYTO(registered trademark)RNASelect(trademark), Hoechst 33258, Quant-iT(trademark)RiboGreen(registered trademark)RNA Reagent, or ethidium bromide, and is preferably SYTO(registered trademark)RNASelect.

[0013] More preferably, the method further includes a step (5) of scaling up verification.

[0014] More preferably, the microbial strains selected in step (4) are subjected to fermentation verification in a fermentation tank, and their RNA content is measured.

[0015] More preferably, the fermentation medium contains, by mass percentage, 2-6% glucose, 0.5-2% yeast extract, 0.2-0.4% magnesium sulfate heptahydrate, 0.2-0.4% zinc sulfate heptahydrate, 0.5-1.2% potassium dihydrogen phosphate, and the remainder being water. More preferably, the pH of the fermentation medium is 5.0 to 6.0. More preferably, the fermentation stirring rotation speed is 300 to 600 rpm. More preferably, the fermentation aeration rate is 3-6 L / min. [Effects of the Invention]

[0016] By employing the method provided in this invention, high RNA yeast strains can be efficiently screened, and dominant strains can be rapidly bred. The RNA content of the screened dominant strains can reach 15% or more, and the wet weight of the cells can reach 200 g / L or more. High-density fermentation of high RNA strains can be achieved, and such strains can reduce the production costs of related products such as RNA and nucleotides in actual production processes. [Brief explanation of the drawing]

[0017] [Figure 1]Figure 1 shows the changes in the OD600 and RNA content of the adaptively evolved bacteria with the inhibitor concentration. Here, (a) shows the changes in the OD600 of the adaptively evolved bacteria, and (b) shows the changes in the RNA content of the adaptively evolved bacteria. [Figure 2] Figure 2 shows the relationship between the RNA content and fluorescence intensity in yeast cells. Here, (A) shows the relationship between the RNA content and fluorescence intensity in yeast cells, and (B) shows the correlation analysis between the RNA content and fluorescence intensity in yeast cells. [Figure 3] Figure 3 shows the cell sorting results by flow cytometry. Here, (a) shows the relationship between the fluorescence intensity and side scatter intensity of the starting strain, (b) shows the relationship between the fluorescence intensity and the number of cells of the starting strain, (c) shows the relationship between the fluorescence intensity and side scatter intensity of the mutagenized evolution library, and (d) shows the relationship between the fluorescence intensity and the number of cells of the mutagenized evolution library. [Figure 4] Figure 4 shows the scale-up verification results of the dominant strain in a 3L fermenter. Here, (a) shows the biomass and RNA results of the strains obtained in Examples 1, 2, and 3 and the starting strain Saccharomyces cerevisiae FX-2 strain in a 3L fermenter, and (b) shows the biomass and RNA results of the strains obtained in Examples 7, 8, and 9 and the starting strain Saccharomyces cerevisiae FX-2 strain in a 3L fermenter.

Mode for Carrying Out the Invention

[0018] The high-throughput breeding method of high-nucleic acid yeast provided by the present invention reflects the RNA content by fluorescence value, adopts the cell sorting technology by flow cytometry, and rapidly breeds dominant strains with high RNA content.

[0019] High-throughput screening is a more efficient method compared to conventional screening. The high-throughput breeding method for high-nucleic acid yeast provided in the present invention involves first staining yeast cells with RNA dyes during a large-scale screening process. After staining, the cells emit fluorescence at specific wavelengths, and fluorescence intensity and RNA content show a positive correlation. Fluorescence analysis is then performed on the stained cells using a flow cytometer, allowing for rapid screening of cells with high fluorescence intensity (i.e., high RNA content).

[0020] In a specific embodiment provided by the present invention, high nucleic acid yeast is screened by a method comprising the following steps.

[0021] (1) Construction of a microbial mutant library: In a specific embodiment of the present invention, a microbial mutant library is obtained by mutagenesis. In a specific embodiment of the present invention, the mutagenesis is chemical mutagenesis, physical mutagenesis, or a combination of chemical and physical mutagenesis.

[0022] In specific embodiments of the present invention, the chemical mutagenic agent is one or more selected from ethyl methanesulfonate, diethyl sulfate, and sodium azide. In a specific embodiment of the present invention, the effective concentration of the chemical mutagenic agent is 1-6% (w / v), and the duration of action is 0.5-2 hours.

[0023] In a specific embodiment of the present invention, the physical mutagenesis method is one or more combinations of ultraviolet radiation and ambient pressure room temperature plasma. In a specific embodiment of the present invention, the time for physical mutagenesis is 30s to 90s.

[0024] (2) Restoration of bacterial cells: In a specific embodiment of the present invention, mutagenic bacterial cells are placed in a culture medium and cultured statically. In a preferred embodiment of the present invention, the culture medium is YPD medium with a pH of 5.0 to 6.0, and the cells are cultured statically at 30°C for 30 to 60 minutes.

[0025] (3) Adaptive evolution of mutant libraries: In a specific embodiment of the present invention, the restored bacterial cells are subcultured in an evolutionary medium containing an inhibitory substance, cultured with shaking, cultured for 20-30 hours, subcultured again, transferred to fresh evolutionary medium and cultured continuously, and the subculture is repeated. Furthermore, the concentration of the inhibitory substance is gradually increased during the subculture process.

[0026] In specific embodiments of the present invention, the inhibitor in the evolutionary culture medium is one or more of the following: 6-azauracil, 8-azaguanine, cycloheximide, 6-mercaptopurine, diaminopurine, 5-bromouracil, and 5-fluorouracil.

[0027] In a specific embodiment of the present invention, the initial concentration of the inhibitory substance is 50 mg to 100 mg / L.

[0028] (4) High-throughput screening of evolutionary libraries: In a specific embodiment of the present invention, a mutagenic library is stained with an RNA-specific fluorescent dye, and after staining, fluorescence analysis is performed on individual cells using a flow cytometer. Strains with high fluorescence intensity (i.e., high RNA content) are selected by utilizing the property that fluorescence intensity and RNA content in yeast cells show a positive correlation.

[0029] (5) Scale-up verification: In a specific embodiment of the present invention, the microbial strain selected in the previous step can be further subjected to fermentation verification in a fermentation tank, and its RNA content can be measured.

[0030] In a specific embodiment of the present invention, the fermentation medium consists of, by mass percentage, 2-6% glucose, 0.5-2% yeast extract, 0.2-0.4% magnesium sulfate heptahydrate, 0.2-0.4% zinc sulfate heptahydrate, 0.5-1.2% potassium dihydrogen phosphate, and the remainder being water, with a pH of 5.0-6.0. In a specific embodiment of the present invention, the mixture is stirred at 300-600 rpm, aerated at 3-6 L / min, and the biomass and RNA content are measured at regular intervals.

[0031] In an embodiment of the present invention, mutagenesis is performed using atmospheric pressure, room temperature plasma, with an output of 100 W and a gas flow rate of 10 L / min. In the embodiment of the present invention, mutagenesis is performed using ultraviolet radiation, with a mutagenesis output of 15W to 20W and an irradiation distance of 30cm.

[0032] The method for detecting biomass and RNA content in the embodiments of the present invention is as follows.

[0033] Biomass detection method: 1. Take a 10 mL centrifuge tube and accurately weigh the empty tube m1. 2. Accurately take 10 mL of fermentation liquid, centrifuge at 5000 rpm for 10 min, discard the supernatant, dry at 105°C until constant weight is reached, and weigh m2. 3. Biomass DCW (g / L) = (m2 - m1) * 100.

[0034] RNA detection methods: Reagent: 1.0.5N perchloric acid (HClO4): Take 400 mL of distilled water, add 21.5 mL of 70% (or 22.1 mL of 68%) perchloric acid, and then add distilled water to bring the total volume to 500 mL. 2.0.25N perchloric acid (HClO4): Take 250 mL of 0.5N perchloric acid (HClO4) and dilute it with distilled water to 500 mL.

[0035] Apparatus: (1) Milligram-class balance, (2) 4000 rpm centrifuge (capable of separating yeast), (3) Centrifuge test tubes (at least 10 mL capacity), (4) Spectrophotometer (260 nm), (5) 70°C water bath, (6) 4°C cold water bath, (7) 10 mL and 1 mL pipette guns, (8) 100 mL volumetric flask.

[0036] method: 1. If using dried yeast powder, weigh 0.06 to 0.15 g of yeast and add it to a centrifuge tube. If using 18% yeast milk, weigh 0.4 to 0.8 g. If using 3.5% liquid, weigh 1.5 to 3.0 g. Add 2.8 mL of cold 0.25 N HClO4 to a centrifuge tube and bathe in water at 4°C for 15 minutes. 3. Centrifuge at 4000 rpm for 10 minutes. 4. Gently discard any floating material on the surface, add 45 mL of 0.5 N HClO, shake to mix uniformly, and bathe in water at 70°C for 15 minutes, shaking once every 3-4 minutes. Centrifuge at 4000 rpm for 10 minutes, aspirate 1 mL of the supernatant, and dilute to 100 mL with distilled water. The absorbance was measured at 5.260 nm, and distilled water was used as a blank control. Calculation formula: %RNA in dried yeast = (absorbance × dilution factor × 0.03365 × 5) / (sample mass × percentage of solid matter in the sample) × 100% In the calculation process for the RNA content in dried yeast according to the present invention, the sample weight is converted to milligrams. For example, if the weight of the weighed dried yeast is 145 mg, the absorbance is 0.7, and the solid content is 96%, the calculation process for the RNA content in the obtained dried yeast is: %RNA = (0.7 × 100 × 0.03365 × 5) / (145 mg × 0.96) × 100%.

[0037] Explanation of related nouns according to the present invention: OD260 / OD600 refers to the RNA content of a single bacterial cell. FV / OD600 refers to the fluorescence intensity of a single bacterial cell (after staining). DCW refers to the dry weight of the bacterial cells.

[0038] The sources of the reagents and equipment used in each of the following examples are shown in Table 1 below.

[0039] [Table 1]

[0040] The Saccharomyces cerevisiae FX-2 strain used in this invention was deposited with the China Center for the Preservation of Typical Cultures (CCTCC) on August 1, 2016, with deposit number CCTCC NO: M2016418. This strain is described in the published patent application with disclosure number CN108220175A.

[0041] (Example 1) Mutagenesis of the starting strain: Saccharomyces cerevisiae FX-2 was cultured in 100 mL of YPD medium at 30°C and 200 rpm for 24 hours with shaking. Then, 1 mL of the bacterial suspension was aspirated, and the cells were collected by centrifugation at 12000 rpm for 1 minute. The cells were washed and precipitated twice with phosphate buffer, and 1 mL of prepared 5% ethyl methanesulfonate (EMS) (containing 5 g of ethyl methanesulfonate per 100 mL of solution) was added. The mixture was homogenized, and the culture was shaken at 30°C and 50 rpm for 30 minutes, followed by centrifugation and washing and precipitation twice. Cell restoration: 5 mL of YPD medium was added to the above cell precipitate to resuspend it, and after static incubation at 30°C for 60 minutes, the cells were collected by centrifugation at 5000 rpm for 5 minutes. Adaptive evolution of the mutagenesis library: 6.7 g of YNB, 20 g of ammonium sulfate, and 100 mg of 6-azauracil were prepared in 1 L of evolution medium solution, filtered through 0.22 μm to remove bacteria, 50 mL of evolution medium was taken, the above bacterial cells were resuspended, and the culture was incubated at 30°C and 200 rpm for 24 hours with shaking. Then, 0.5 mL of the culture solution was aspirated and transferred to fresh evolution medium and cultured for another 24 hours. This process was repeated 50 times for subculturing, and the concentration of the inhibitory substance was gradually increased to 200, 300, 400, and 500 mg / L. After evolution was complete, the bacterial suspension obtained from the final culture was collected, and the bacterial cells were collected by centrifugation. Figure 1 shows the changes in the OD600 and RNA content of the obtained bacterial cells in relation to the concentration of the inhibitory substance. Here, (a) shows the change in adaptive evolution bacterial cell OD600, and (b) shows the change in adaptive evolution RNA content. As can be seen from Figure 1, the bacterial OD600 value and RNA content increase as the concentration of the inhibitory substance increases. High-throughput screening of evolutionary libraries: SYTO (registered trademark) RNASelect (trademark) was diluted 10,000-fold to prepare a 500 nM staining solution. 1 mL was aspirated and added to the cells obtained by centrifugation after evolution. The cells were resuspended, incubated at 30°C and 50 rpm for 20 minutes with shaking, and then centrifuged at 5000 rpm for 5 minutes to collect the cells. The cells were washed twice with phosphate buffer, precipitated, and resuspended. Fluorescence analysis was performed on the resuspended cells using a flow cytometer, with selection conditions: excitation wavelength 490 nm, emission wavelength 530 nm. The flow cytometer analysis showed that in the evolved cell library, the fluorescence of one cell cluster was significantly higher than that of other cells. Figure 2 shows the relationship between RNA content in yeast cells and fluorescence intensity. Here, (a) shows the relationship between RNA content in yeast cells and fluorescence intensity, and (b) shows the correlation analysis between RNA content in yeast cells and fluorescence intensity. As can be seen from Figure 2, the RNA content in yeast cells shows a linear correlation with fluorescence intensity. Figure 3 shows the results of cell sorting by flow cytometry. Here, (a) shows the relationship between the fluorescence intensity and lateral scattering intensity of the starting strain, (b) shows the relationship between the fluorescence intensity and cell number of the starting strain, (c) shows the relationship between the fluorescence intensity and lateral scattering intensity of the mutagenic evolution library, and (d) shows the relationship between the fluorescence intensity and cell number of the mutagenic evolution library. As can be seen from Figure 3, when the evolved cell library was sorted by flow cytometry, the fluorescence of one cell cluster was clearly higher than that of the other cells. Scale-up verification: The dominant strain A, screened using the flow cytometer described above, underwent fermentation verification in a 3L fermenter. The fermentation medium consisted of 6% glucose, 2% yeast extract, 0.4% magnesium sulfate heptahydrate, 0.4% zinc sulfate heptahydrate, 1.2% potassium dihydrogen phosphate, and the remainder water, expressed by mass percentage. The pH was 5.5, the medium was stirred at 600 rpm, aerated at 4 L / min, and the biomass and RNA content were measured at regular intervals. Upon completion of fermentation, the RNA content reached 15.2%, and the dry weight of the bacterial cells reached 51.7 g / L. Figure 4 shows the results of the scale-up verification of dominant strain A in a 3L fermenter. As can be seen from Figure 4, the RNA content obtained after fermentation culture of the screened dominant strain A in a 3L fermenter is clearly higher than that of the starting strain Saccharomyces cerevisiae FX-2.

[0042] (Example 2) Mutagenesis of the starting strain: Saccharomyces cerevisiae FX-2 was cultured in 100 mL of YPD medium at 30°C and 200 rpm for 24 hours with shaking. Then, 1 mL of the bacterial suspension was aspirated, and the cells were collected by centrifugation at 12000 rpm for 1 minute. The cells were washed and precipitated twice with phosphate buffer, and 1 mL of prepared 3% sodium azide (containing 3 g of sodium azide per 100 mL of solution) was added. The mixture was homogenized, and after shaking at 30°C and 50 rpm for 60 minutes, it was centrifuged and washed and precipitated twice. Cell restoration: 5 mL of YPD medium was added to the above cell precipitate to resuspend it, and after static incubation at 30°C for 60 minutes, the cells were collected by centrifugation at 5000 rpm for 5 minutes. Adaptive evolution of the mutagenesis library: 6.7 g of YNB, 20 g of ammonium sulfate, and 50 mg of 8-azaguanine were prepared in 1 L of evolution medium solution, filtered through 0.22 μm to remove bacteria, 50 mL of evolution medium was taken, the above bacterial cells were resuspended, and the culture was incubated at 30°C and 200 rpm for 24 hours with shaking. Then, 0.5 mL of the culture solution was aspirated and transferred to fresh evolution medium and cultured for another 24 hours. This process was repeated 100 times for subculturing, and the concentration of the inhibitory substance was gradually increased to 100, 150, 200, 250, and 300 mg / L. After evolution was complete, the bacterial suspension obtained from the final culture was collected, and the bacterial cells were collected by centrifugation. High-throughput screening of evolutionary libraries: Quant-iT® RiboGreen® RNA Reagent was diluted in a 500 nM staining solution, 1 mL was aspirated, and added to the cells obtained by centrifugation after evolution. The cells were then resuspended, incubated with shaking at 30°C and 50 rpm for 20 min, and then centrifuged at 5000 rpm for 5 min to collect the cells. The cells were washed twice with phosphate buffer, precipitated, and resuspended. Fluorescence analysis was performed on the resuspended cells using a flow cytometer, with selection conditions: excitation wavelength 490 nm, emission wavelength 530 nm. Analysis by flow cytometer showed that in the evolved cell library, the fluorescence of one cell cluster was significantly higher than that of other cells. Scale-up verification: The dominant strain B screened using the flow cytometer described above was subjected to fermentation verification in a 3L fermenter. The fermentation medium consisted of 2% glucose, 0.5% yeast extract, 0.4% magnesium sulfate heptahydrate, 0.4% zinc sulfate heptahydrate, 1.2% potassium dihydrogen phosphate, and the remainder water, expressed by mass percentage. The pH was 6.0, and the medium was stirred at 400 rpm and aerated at 4 L / min. The biomass and RNA content were measured at regular intervals. Upon completion of fermentation, the RNA content reached 15.1%, and the dry weight of the bacterial cells reached 53.2 g / L. Figure 4 shows the results of the scale-up verification of dominant strain B in a 3L fermenter. As can be seen from Figure 4, the RNA content obtained after fermentation culture of the screened dominant strain B in a 3L fermenter is clearly higher than that of the starting strain Saccharomyces cerevisiae FX-2.

[0043] (Example 3) Mutagenesis of the starting strain: Saccharomyces cerevisiae FX-2 was cultured in 100 mL of YPD medium with shaking at 30°C and 200 rpm for 24 hours. Then, 1 mL of the bacterial suspension was aspirated, and the cells were collected by centrifugation at 12000 rpm for 1 minute. The cells were washed and precipitated twice with phosphate buffer, and 1 mL of prepared 3% diethyl sulfate (containing 3 g of diethyl sulfate per 100 mL of solution) was added and mixed uniformly. After shaking at 30°C and 50 rpm for 60 minutes, mutagenesis was induced for 60 seconds under atmospheric pressure room temperature plasma (ARTP) conditions, followed by centrifugation and washing and precipitation twice. Cell restoration: 5 mL of YPD medium was added to the above cell precipitate to resuspend it, and after static incubation at 30°C for 60 minutes, the cells were collected by centrifugation at 5000 rpm for 5 minutes. Adaptive evolution of the mutagenesis library: 6.7 g of YNB, 20 g of ammonium sulfate, and 50 mg of cycloheximide were prepared in 1 L of evolution medium solution, filtered through 0.22 μm to remove bacteria, 50 mL of evolution medium was taken, the above bacterial cells were resuspended, and the culture was incubated at 30°C and 200 rpm for 24 hours with shaking. Then, 0.5 mL of the culture solution was aspirated and transferred to fresh evolution medium and cultured for another 24 hours. This process was repeated 150 times for subculturing, and the concentration of the inhibitor was gradually increased to 200, 300, 400, and 500 mg / L. After evolution was complete, the bacterial suspension obtained from the final culture was collected, and the bacterial cells were collected by centrifugation. High-throughput screening of evolutionary libraries: SYTO (registered trademark) RNASelect (trademark) was diluted 10,000-fold to prepare a 500 nM staining solution. 1 mL was aspirated and added to the cells obtained by centrifugation after evolution. The cells were resuspended, incubated at 30°C and 50 rpm for 20 minutes with shaking, and then centrifuged at 5000 rpm for 5 minutes to collect the cells. The cells were washed twice with phosphate buffer, precipitated, and resuspended. Fluorescence analysis was performed on the resuspended cells using a flow cytometer, with selection conditions: excitation wavelength 490 nm, emission wavelength 530 nm. The flow cytometer analysis showed that in the evolved cell library, the fluorescence of one cell cluster was significantly higher than that of other cells. Scale-up verification: The dominant strain C screened using the flow cytometer described above was subjected to fermentation verification in a 3L fermenter. The fermentation medium consisted of 4% glucose, 1% yeast extract, 0.2% magnesium sulfate heptahydrate, 0.2% zinc sulfate heptahydrate, 1% potassium dihydrogen phosphate, and the remainder water, expressed by mass percentage. The pH was 5.0, the medium was stirred at 600 rpm, aerated at 6 L / min, and the biomass and RNA content were measured at regular intervals. Upon completion of fermentation, the RNA content reached 15.5%, and the dry weight of the bacterial cells reached 37.1 g / L. Figure 4 shows the results of the scale-up verification of dominant strain C in a 3L fermenter. As can be seen from Figure 4, the RNA content obtained after fermentation culture of the screened dominant strain C in a 3L fermenter is clearly higher than that of the starting strain Saccharomyces cerevisiae FX-2.

[0044] (Example 4) Mutagenesis of the starting strain: Saccharomyces cerevisiae FX-2 was cultured in 100 mL of YPD medium with shaking at 30°C and 200 rpm for 24 hours. Then, 1 mL of the bacterial suspension was aspirated, and the cells were collected by centrifugation at 12000 rpm for 1 minute. The cells were washed and precipitated twice with phosphate buffer, and 1 mL of prepared 5% ethyl methanesulfonate (containing 5 g of ethyl methanesulfonate per 100 mL of solution) was added and mixed uniformly. After shaking at 30°C and 50 rpm for 30 minutes, mutagenesis was induced for 30 seconds under ultraviolet radiation, followed by centrifugation and washing and precipitation twice. Cell restoration: 5 mL of YPD medium was added to the above cell precipitate to resuspend it, and after static incubation at 30°C for 60 minutes, the cells were collected by centrifugation at 5000 rpm for 5 minutes. Adaptive evolution of the mutagenesis library: 6.7 g of YNB, 20 g of ammonium sulfate, and 50 mg of 6-mercaptopurine were prepared in 1 L of evolution medium solution, filtered through 0.22 μm to remove bacteria, 50 mL of evolution medium was taken, the above bacterial cells were resuspended, and the culture was incubated at 30°C and 200 rpm for 24 hours with shaking. Then, 0.5 mL of the culture solution was aspirated and transferred to fresh evolution medium and cultured for another 24 hours. This process was repeated 60 times for subculturing, and the concentration of the inhibitor was gradually increased to 100, 150, 200, 250, and 300 mg / L. After evolution was complete, the bacterial suspension obtained from the final culture was collected, and the bacterial cells were collected by centrifugation. High-throughput screening of the evolutionary library: Dilute ethidium bromide in a 1 ug / mL staining solution, aspirate 1 mL, add it to the cells obtained by centrifugation after evolution, resuspend the cells, incubate with shaking at 30°C and 50 rpm for 20 min, collect the cells by centrifugation at 5000 rpm for 5 min, wash and precipitate twice with phosphate buffer, resuspend, and perform fluorescence analysis of the resuspended suspension using a flow cytometer. Selection conditions: excitation wavelength 490 nm, emission wavelength 530 nm. Analysis by flow cytometer showed that in the evolved cell library, the fluorescence of one cell cluster was significantly higher than that of other cells. Scale-up verification: The dominant bacterial strains screened using the flow cytometer described above were subjected to fermentation verification in a 3L fermenter. The fermentation medium consisted of 2% glucose, 0.5% yeast extract, 0.2% magnesium sulfate heptahydrate, 0.2% zinc sulfate heptahydrate, 0.5% potassium dihydrogen phosphate, and the remainder water, expressed by mass percentage. The pH was 5.0, the medium was stirred at 300 rpm, aerated at 3 L / min, and the biomass and RNA content were measured at regular intervals. Upon completion of fermentation, the RNA content reached 15.3%, and the dry weight of the bacterial cells reached 52.9 g / L.

[0045] (Example 5) Mutagenesis of the starting strain: Saccharomyces cerevisiae FX-2 was cultured in 100 mL of YPD medium at 30°C and 200 rpm for 24 hours with shaking. Then, 1 mL of the bacterial suspension was aspirated, and the cells were collected by centrifugation at 12000 rpm for 1 minute. The cells were washed and precipitated twice with phosphate buffer, and 1 mL of prepared 5% ethyl methanesulfonate (EMS) (containing 5 g of ethyl methanesulfonate per 100 mL of solution) was added. The mixture was homogenized, and the culture was shaken at 30°C and 50 rpm for 30 minutes, followed by centrifugation and washing and precipitation twice. Cell restoration: 5 mL of YPD medium was added to the above cell precipitate to resuspend it, and after static incubation at 30°C for 60 minutes, the cells were collected by centrifugation at 5000 rpm for 5 minutes. Adaptive evolution of the mutagenesis library: 6.7 g of YNB, 20 g of ammonium sulfate, and 100 mg of diaminopurine were prepared in 1 L of evolution medium solution, filtered through 0.22 μm to remove bacteria, 50 mL of evolution medium was taken, the above bacterial cells were resuspended, and the culture was incubated at 30°C and 200 rpm for 24 hours with shaking. Then, 0.5 mL of the culture solution was aspirated and transferred to fresh evolution medium and cultured for another 24 hours. This process was repeated 120 times for subculturing, and the concentration of the inhibitory substance was gradually increased to 200, 300, 400, and 500 mg / L. After evolution was complete, the bacterial suspension obtained from the final culture was collected, and the bacterial cells were collected by centrifugation. High-throughput screening of evolutionary libraries: Hoechst 33258 was diluted in 1 μM staining solution, 1 mL was aspirated, and added to the cells obtained by centrifugation after evolution. The cells were resuspended, incubated with shaking at 30°C and 50 rpm for 20 min, and then centrifuged at 5000 rpm for 5 min to collect the cells. The cells were washed twice with phosphate buffer, precipitated, and resuspended. Fluorescence analysis was performed on the resuspended suspension using a flow cytometer, with selection conditions: excitation wavelength 490 nm, emission wavelength 530 nm. Analysis by flow cytometer showed that in the evolved cell library, the fluorescence of one cell cluster was significantly higher than that of other cells. Scale-up verification: The dominant bacterial strains screened using the flow cytometer described above were subjected to fermentation verification in a 3L fermenter. The fermentation medium consisted of 6% glucose, 2% yeast extract, 0.4% magnesium sulfate heptahydrate, 0.4% zinc sulfate heptahydrate, 1.2% potassium dihydrogen phosphate, and the remainder water, expressed by mass percentage. The pH was 5.5, the medium was stirred at 600 rpm, aerated at 4 L / min, and the biomass and RNA content were measured at regular intervals. Upon completion of fermentation, the RNA content reached 15.2%, and the dry weight of the bacterial cells reached 51.7 g / L.

[0046] (Example 6) Mutagenesis of the starting strain: Saccharomyces cerevisiae FX-2 was cultured in 100 mL of YPD medium at 30°C and 200 rpm for 24 hours with shaking. Then, 1 mL of the bacterial suspension was aspirated, and the cells were collected by centrifugation at 12000 rpm for 1 minute. The cells were washed and precipitated twice with phosphate buffer, and 1 mL of prepared 3% sodium azide (containing 3 g of sodium azide per 100 mL of solution) was added. The mixture was homogenized, and after shaking at 30°C and 50 rpm for 60 minutes, it was centrifuged and washed and precipitated twice. Cell restoration: 5 mL of YPD medium was added to the above cell precipitate to resuspend it, and after static incubation at 30°C for 60 minutes, the cells were collected by centrifugation at 5000 rpm for 5 minutes. Adaptive evolution of the mutagenesis library: 6.7 g of YNB, 20 g of ammonium sulfate, and 50 mg of 5-bromouracil were prepared in 1 L of evolution medium solution, filtered through 0.22 μm to remove bacteria, 50 mL of evolution medium was taken, the above bacterial cells were resuspended, and the culture was incubated at 30°C and 200 rpm for 24 hours with shaking. Then, 0.5 mL of the culture solution was aspirated and transferred to fresh evolution medium and incubated for another 24 hours. This process was repeated 90 times for subculturing, and the concentration of the inhibitor was gradually increased to 100, 150, 200, 250, and 300 mg / L. After evolution was complete, the bacterial suspension obtained from the final culture was collected, and the bacterial cells were collected by centrifugation. High-throughput screening of evolutionary libraries: Quant-iT® RiboGreen® RNA Reagent was diluted in 500 nM staining solution, 1 mL was aspirated, and added to the cells obtained by centrifugation after evolution. The cells were resuspended, incubated with shaking at 30°C and 50 rpm for 20 min, and then centrifuged at 5000 rpm for 5 min to collect the cells. The cells were washed twice with phosphate buffer, precipitated, and resuspended. Fluorescence analysis was performed on the resuspended cells using a flow cytometer, with selection conditions: excitation wavelength 490 nm, emission wavelength 530 nm. Analysis by flow cytometer showed that in the evolved cell library, the fluorescence of one cell cluster was significantly higher than that of other cells. Scale-up Verification: The dominant bacterial strains screened using the flow cytometer described above were subjected to fermentation verification in a 3L fermenter. The fermentation medium consisted of 2% glucose, 0.5% yeast extract, 0.4% magnesium sulfate heptahydrate, 0.4% zinc sulfate heptahydrate, 1.2% potassium dihydrogen phosphate, and the remainder water, expressed by mass percentage. The pH was 6.0, the medium was stirred at 400 rpm, aerated at 4 L / min, and the biomass and RNA content were measured at regular intervals. Upon completion of fermentation, the RNA content reached 15.4%, and the dry weight of the bacterial cells reached 53.2 g / L.

[0047] (Example 7) Mutagenesis of starting strains: Saccharomyces cerevisiae FX-2 was cultured in 100 mL of YPD medium with shaking at 30°C and 200 rpm for 24 hours. Then, 1 mL of the bacterial suspension was aspirated, and the cells were collected by centrifugation at 12000 rpm for 1 minute. The cells were washed and precipitated twice with phosphate buffer, and then mutagenesis was induced for 60 seconds under atmospheric pressure room temperature plasma (ARTP) conditions, followed by centrifugation and washing and precipitation twice. Cell restoration: 5 mL of YPD medium was added to the above cell precipitate to resuspend it, and after static incubation at 30°C for 60 minutes, the cells were collected by centrifugation at 5000 rpm for 5 minutes. Adaptive evolution of the mutagenesis library: 6.7 g of YNB, 20 g of ammonium sulfate, and 50 mg of 5-fluorouracil were prepared in 1 L of evolution medium solution, filtered through 0.22 μm to remove bacteria, 50 mL of evolution medium was taken, the above bacterial cells were resuspended, and the culture was incubated at 30°C and 200 rpm for 24 hours with shaking. Then, 0.5 mL of the culture solution was aspirated and transferred to fresh evolution medium and cultured for another 24 hours. This process was repeated 130 times for subculturing, and the concentration of the inhibitor was gradually increased to 100, 150, 200, 250, and 300 mg / L. After evolution was complete, the bacterial suspension obtained from the final culture was collected, and the bacterial cells were collected by centrifugation. High-throughput screening of the evolutionary library: Dilute ethidium bromide in a 1 ug / mL staining solution, aspirate 1 mL, add it to the cells obtained by centrifugation after evolution, resuspend the cells, incubate with shaking at 30°C and 50 rpm for 20 min, collect the cells by centrifugation at 5000 rpm for 5 min, wash and precipitate twice with phosphate buffer, resuspend, and perform fluorescence analysis of the resuspended suspension using a flow cytometer. Selection conditions: excitation wavelength 490 nm, emission wavelength 530 nm. Analysis by flow cytometer showed that in the evolved cell library, the fluorescence of one cell cluster was significantly higher than that of other cells. Scale-up Verification: The dominant strain D screened using the flow cytometer described above was subjected to fermentation verification in a 3L fermenter. The fermentation medium consisted of 6% glucose, 2% yeast extract, 0.2% magnesium sulfate heptahydrate, 0.2% zinc sulfate heptahydrate, 1% potassium dihydrogen phosphate, and the remainder water, expressed by mass percentage. The pH was 5.5, the medium was stirred at 400 rpm, aerated at 3 L / min, and the biomass and RNA content were measured at regular intervals. Upon completion of fermentation, the RNA content reached 15.0%, and the dry weight of the bacterial cells reached 53.5 g / L. Figure 4 shows the results of the scale-up verification of dominant strain D in a 3L fermenter. As can be seen from Figure 4, the RNA content obtained after fermentation culture of the screened dominant strain D in a 3L fermenter is clearly higher than that of the starting strain Saccharomyces cerevisiae FX-2.

[0048] (Example 8) Mutagenesis of the starting strain: Saccharomyces cerevisiae FX-2 was cultured in 100 mL of YPD medium with shaking at 30°C and 200 rpm for 24 hours. Then, 1 mL of the bacterial suspension was aspirated, and the cells were collected by centrifugation at 12000 rpm for 1 minute. The cells were washed and precipitated twice with phosphate buffer, and 1 mL of prepared 3% diethyl sulfate (containing 3 g of diethyl sulfate per 100 mL of solution) was added and mixed uniformly. After shaking at 30°C and 50 rpm for 60 minutes, mutagenesis was induced for 60 seconds under atmospheric pressure room temperature plasma (ARTP) conditions, followed by centrifugation and washing and precipitation twice. Cell restoration: 5 mL of YPD medium was added to the above cell precipitate to resuspend it, and after static incubation at 30°C for 60 minutes, the cells were collected by centrifugation at 5000 rpm for 5 minutes. Adaptive evolution of the mutagenesis library: 6.7 g of YNB, 20 g of ammonium sulfate, 25 mg of 8-azaguanine, and 25 mg of cycloheximide were prepared in 1 L of evolution medium solution, filtered through 0.22 μm to remove bacteria, 50 mL of evolution medium was taken, the above bacterial cells were resuspended, and the culture was incubated at 30°C and 200 rpm for 24 hours with shaking. Then, 0.5 mL of the culture solution was aspirated and transferred to fresh evolution medium and incubated for another 24 hours. This process was repeated 50 times for subculturing, and the total concentration of the inhibitory substance was gradually increased to 100, 150, 200, 250, and 300 mg / L. After evolution was complete, the bacterial suspension obtained from the final culture was collected, and the bacterial cells were collected by centrifugation. High-throughput screening of evolutionary libraries: Hoechst 33258 was diluted in 1 μM staining solution, 1 mL was aspirated, and added to the cells obtained by centrifugation after evolution. The cells were resuspended, incubated with shaking at 30°C and 50 rpm for 20 min, and then centrifuged at 5000 rpm for 5 min to collect the cells. The cells were washed twice with phosphate buffer, precipitated, and resuspended. Fluorescence analysis was performed on the resuspended suspension using a flow cytometer, with selection conditions: excitation wavelength 490 nm, emission wavelength 530 nm. Analysis by flow cytometer showed that in the evolved cell library, the fluorescence of one cell cluster was significantly higher than that of other cells. Scale-up verification: The dominant strain E, screened using the flow cytometer described above, underwent fermentation verification in a 3L fermenter. The fermentation medium consisted of 4% glucose, 1% yeast extract, 0.2% magnesium sulfate heptahydrate, 0.2% zinc sulfate heptahydrate, 1% potassium dihydrogen phosphate, and the remainder water, expressed by mass percentage. The pH was 5.0, the medium was stirred at 600 rpm, aerated at 6 L / min, and the biomass and RNA content were measured at regular intervals. Upon completion of fermentation, the RNA content reached 15.4%, and the dry weight of the microbial cells reached 52.6 g / L. Figure 4 shows the results of the scale-up verification of dominant strain E in a 3L fermenter. As can be seen from Figure 4, the RNA content obtained after fermentation culture of the screened dominant strain E in a 3L fermenter is clearly higher than that of the starting strain Saccharomyces cerevisiae FX-2.

[0049] (Example 9) Mutagenesis of the starting strain: Saccharomyces cerevisiae FX-2 was cultured in 100 mL of YPD medium with shaking at 30°C and 200 rpm for 24 hours. Then, 1 mL of the bacterial suspension was aspirated, and the cells were collected by centrifugation at 12000 rpm for 1 minute. The cells were washed and precipitated twice with phosphate buffer, and 1 mL of prepared 5% ethyl methanesulfonate (containing 5 g of ethyl methanesulfonate per 100 mL of solution) was added and mixed uniformly. After shaking at 30°C and 50 rpm for 30 minutes, mutagenesis was induced for 30 seconds under ultraviolet radiation, followed by centrifugation and washing and precipitation twice. Cell restoration: 5 mL of YPD medium was added to the above cell precipitate to resuspend it, and after static incubation at 30°C for 60 minutes, the cells were collected by centrifugation at 5000 rpm for 5 minutes. Adaptive evolution of the mutagenesis library: 6.7 g of YNB, 20 g of ammonium sulfate, 40 mg of 5-fluorouracil, and 40 mg of 6-mercaptopurine were prepared in 1 L of evolution medium solution, filtered through 0.22 μm to remove bacteria, 50 mL of evolution medium was taken, the above bacterial cells were resuspended, and the culture was incubated at 30°C and 200 rpm for 24 hours with shaking. Then, 0.5 mL of the culture solution was aspirated and transferred to fresh evolution medium and cultured for another 24 hours. This process was repeated 100 times for subculturing, and the concentration of the inhibitory substance was gradually increased to 100, 200, 300, 400, and 500 mg / L. After evolution was complete, the bacterial suspension obtained from the final culture was collected, and the bacterial cells were collected by centrifugation. High-throughput screening of evolutionary libraries: SYTO (registered trademark) RNASelect (trademark) was diluted 10,000-fold to prepare a 500 nM staining solution. 1 mL was aspirated and added to the cells obtained by centrifugation after evolution. The cells were resuspended, incubated at 30°C and 50 rpm for 20 minutes with shaking, and then centrifuged at 5000 rpm for 5 minutes to collect the cells. The cells were washed twice with phosphate buffer, precipitated, and resuspended. Fluorescence analysis was performed on the resuspended cells using a flow cytometer, with selection conditions: excitation wavelength 490 nm, emission wavelength 530 nm. The flow cytometer analysis showed that in the evolved cell library, the fluorescence of one cell cluster was significantly higher than that of other cells. Scale-up verification: The dominant strain F screened using the flow cytometer described above was subjected to fermentation verification in a 3L fermenter. The fermentation medium consisted of 2% glucose, 0.5% yeast extract, 0.2% magnesium sulfate heptahydrate, 0.2% zinc sulfate heptahydrate, 0.5% potassium dihydrogen phosphate, and the remainder water, expressed by mass percentage. The pH was 5.0, and the medium was stirred at 300 rpm and aerated at 3 L / min. The biomass and RNA content were measured at regular intervals. Upon completion of fermentation, the RNA content reached 15.7%, and the dry weight of the bacterial cells reached 52.9 g / L. Figure 4 shows the results of the scale-up verification of dominant strain F in a 3L fermenter. As can be seen from Figure 4, the RNA content obtained after fermentation culture of the screened dominant strain F in a 3L fermenter is clearly higher than that of the starting strain Saccharomyces cerevisiae FX-2.

[0050] (Comparative Example 1) 1) Mutagenesis of the starting strain: Saccharomyces cerevisiae FX-2 was cultured in 100 mL of YPD medium at 30°C and 200 rpm for 24 hours with shaking. Then, 1 mL of the bacterial suspension was aspirated, and the cells were collected by centrifugation at 12000 rpm for 1 minute. The cells were washed and precipitated twice with phosphate buffer, and 1 mL of prepared 5% ethyl methanesulfonate (EMS) (containing 5 g of ethyl methanesulfonate per 100 mL of solution) was added. The mixture was homogenized, and after shaking at 30°C and 50 rpm for 30 minutes, it was centrifuged and washed and precipitated twice. 2) Restoration of bacterial cells: 5 mL of YPD medium was added to the bacterial cell precipitate and resuspended. After resting culture at 30°C for 60 minutes, the cells were collected by centrifugation at 5000 rpm for 5 minutes. 3) High-throughput screening of evolutionary libraries: Prepare a 500 nM staining solution by diluting SYTO (registered trademark) RNASelect (trademark) 10,000 times, aspirate 1 mL, add it to the bacterial cells obtained by centrifugation after evolution as described above, resuspend the bacterial cells, incubate with shaking at 30°C and 50 rpm for 20 min, collect the bacterial cells by centrifugation at 5000 rpm for 5 min, wash and precipitate twice with phosphate buffer, resuspend, and perform fluorescence analysis of the resuspended suspension using a flow cytometer. Selection conditions: excitation wavelength 490 nm, emission wavelength 530 nm. 4) Scale-up verification: The dominant bacterial strains screened using the flow cytometer described above were subjected to fermentation verification in a 3L fermenter. The fermentation medium consisted of 6% glucose, 2% yeast extract, 0.4% magnesium sulfate heptahydrate, 0.4% zinc sulfate heptahydrate, 1.2% potassium dihydrogen phosphate, and the remainder water, expressed by mass percentage. The pH was 5.5, the medium was stirred at 600 rpm, aerated at 4 L / min, and the biomass and RNA content were measured at regular intervals. Upon completion of fermentation, the RNA content reached 11.8%, and the dry weight of the bacterial cells reached 52.3 g / L.

[0051] (Comparative Example 2) 1) Mutagenesis of the starting strain: Saccharomyces cerevisiae FX-2 was cultured in 100 mL of YPD medium at 30°C and 200 rpm for 24 hours with shaking. Then, 1 mL of the bacterial suspension was aspirated, and the cells were collected by centrifugation at 12000 rpm for 1 minute. The cells were washed and precipitated twice with phosphate buffer, and 1 mL of prepared 5% ethyl methanesulfonate (EMS) (containing 5 g of ethyl methanesulfonate per 100 mL of solution) was added. The mixture was homogenized, and after shaking at 30°C and 50 rpm for 30 minutes, it was centrifuged and washed and precipitated twice. 2) Restoration of bacterial cells: 5 mL of YPD medium was added to the bacterial cell precipitate and resuspended. After resting culture at 30°C for 60 minutes, the cells were collected by centrifugation at 5000 rpm for 5 minutes. 3) Adaptive evolution of the mutagenesis library: 6.7 g of YNB, 20 g of ammonium sulfate, and 100 mg of 6-azauracil were prepared in 1 L of evolution medium solution, filtered through 0.22 μm to remove bacteria, 50 mL of evolution medium was taken, the above bacterial cells were resuspended, and the culture was incubated at 30°C and 200 rpm for 24 hours with shaking. Then, 0.5 mL of the culture solution was aspirated and transferred to fresh evolution medium and cultured for another 24 hours. This process was repeated 50 times for subculturing, and the concentration of the inhibitor was gradually increased to 200, 300, 400, and 500 mg / L. After evolution was complete, the bacterial suspension obtained from the final culture was collected, and the bacterial cells were collected by centrifugation. 4) Screening of the evolutionary library: After evolution as described above, the cells obtained by centrifugation were resuspended, incubated with shaking at 30°C and 50 rpm for 20 minutes, and then centrifuged at 5000 rpm for 5 minutes to collect the cells. The cells were washed twice with phosphate buffer, precipitated, and resuspended. The resuspended cells were diluted to an appropriate concentration and spread onto a YPD solid plate. After static incubation at 30°C for 24 hours, single colonies were picked and the RNA content was measured one by one. 5) Scale-up verification: A strain with high RNA content was selected and fermentation verification was performed in a 3L fermenter. The fermentation medium consisted of 6% glucose, 2% yeast extract, 0.4% magnesium sulfate heptahydrate, 0.4% zinc sulfate heptahydrate, 1.2% potassium dihydrogen phosphate, and the remainder water, expressed by mass percentage. The pH was 5.5, and the medium was stirred at 600 rpm and aerated at 4 L / min. The biomass and RNA content were measured at regular intervals. After fermentation was complete, the RNA content reached 12.6%, and the dry weight of the microbial cells reached 51.6 g / L.

[0052] The foregoing are merely preferred embodiments for carrying out the present invention and do not limit the present invention in any way. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention are all within the scope of protection of the present invention.

[0053] (Note) (Note 1) A high-throughput breeding method for high-ribonucleic acid yeast, Step (1) to construct a microbial mutant library of yeast mutant cells, Step (2) involves restoring the bacterial cells obtained in step (1), Step (3) of adaptively evolving the mutant library, A high-throughput breeding method for high-ribonucleic acid yeast, characterized by comprising the step (4) of obtaining high-RNA yeast by high-throughput screening of an adaptively evolved library.

[0054] (Note 2) The method according to Appendix 1, characterized in that, in step (1), the step of constructing the microbial mutant library employs chemical mutagenesis, physical mutagenesis, or a combination of chemical mutagenesis and physical mutagenesis.

[0055] (Note 3) The chemical mutagenesis agent is one or more of the following: ethyl methanesulfonate, diethyl sulfate, and sodium azide. Preferably, the effective concentration of the chemical mutagenic agent is 1-6% (w / v), The method according to Appendix 2, more preferably characterized in that the duration of action is 0.5 to 2 hours.

[0056] (Note 4) Physical mutagenesis employs one or more combinations of ultraviolet radiation and ambient pressure room-temperature plasma, and preferably the duration of physical mutagenesis is 30s to 90s. Preferably, the output power of the ambient pressure, room temperature plasma is 80-120 W and the gas flow rate is 8-15 L / min, more preferably, the output power of the ambient pressure, room temperature plasma is 100 W and the gas flow rate is 10 L / min. More preferably, the method according to Appendix 2, characterized in that the output of ultraviolet radiation is 15 w to 20 watts and the irradiation distance is 20 to 30 cm.

[0057] (Note 5) In step (2), the bacterial cells are cultured statically in a culture medium. Preferably, the culture medium is YPD medium. More preferably, the pH of the culture medium is 5.0 to 6.0. More preferably, the culture temperature is 28-32°C. More preferably, the method according to any one of the appendices 1 to 4, characterized in that the culture time is 30 to 60 minutes.

[0058] (Note 6) In step (3), the restored bacterial cells obtained in step (2) are cultured in an evolutionary medium containing an inhibitory substance, and the culture solution is collected and subcultured into a new evolutionary medium, and this process is repeated 50 to 150 times. Preferably, the inhibitory substance is one or more of the following: 6-azauracil, 8-azaguanine, cycloheximide, 6-mercaptopurine, diaminopurine, 5-bromouracil, 5-fluorouracil. More preferably, the concentration of inhibitory substances in the culture medium after subculturing increases. More preferably, during the subculturing process, the concentration of the inhibitory substance in the culture medium increases by 50 to 1000 mg / L. More preferably, the culture is performed for 20-30 hours before subculturing, preferably for 24 hours. More preferably, the culture temperature is 28-33°C, and the culture is performed with shaking at 150-220 rpm. More preferably, the concentration of the inhibitory substance is 50 mg to 1000 mg / L, and more preferably, the concentration of the inhibitory substance is 50 mg to 500 mg / L. More preferably, the method according to any one of the appendices 1 to 5, characterized in that the initial concentration of the inhibitory substance is 50 mg to 100 mg / L.

[0059] (Note 7) In step (4), the mutagenesis library is stained with an RNA fluorescent dye, and strains with high fluorescence intensity are selected to obtain high RNA yeast. Preferably, the RNA fluorescent dye is one or more of SYTO(registered trademark)RNASelect(trademark), Hoechst 33258, Quant-iT(trademark)RiboGreen(registered trademark)RNA Reagent, or ethidium bromide, and preferably SYTO(registered trademark)RNASelect, characterized in that the method according to any one of the appendices 1 to 6.

[0060] (Note 8) The method according to any one of the appendices 1 to 7, further comprising step (5) of scaling up verification.

[0061] (Note 9) The method according to Appendix 8, characterized in that, in step (5), a fermentation verification is performed on the bacterial strain selected in step (4) in a fermenter and its RNA content is measured.

[0062] (Note 10) The fermentation medium, expressed by mass percentage, contains 2-6% glucose, 0.5-2% yeast extract, 0.2-0.4% magnesium sulfate heptahydrate, 0.2-0.4% zinc sulfate heptahydrate, 0.5-1.2% potassium dihydrogen phosphate, and the remainder is water. Preferably, the pH of the fermentation medium is 5.0 to 6.0. More preferably, the fermentation stirring rotation speed is 300 to 600 rpm. More preferably, the method according to Appendix 9, characterized in that the fermentation aeration rate is 3 to 6 L / min.

Claims

1. A high-throughput breeding method for high-ribonucleic acid yeast, Step (1) to construct a microbial mutant library of yeast mutant cells, Step (2) involves restoring the bacterial cells obtained in step (1), Step (3) of adaptively evolving the mutant library, A high-throughput breeding method for high-ribonucleic acid yeast, characterized by comprising the step (4) of obtaining high-RNA yeast by high-throughput screening of an adaptively evolved library.

2. The method according to claim 1, characterized in that, in step (1), the step of constructing the microbial mutant library employs chemical mutagenesis, physical mutagenesis, or a combination of chemical mutagenesis and physical mutagenesis.

3. The chemical mutagenesis agent is one or more of the following: ethyl methanesulfonate, diethyl sulfate, and sodium azide. Preferably, the effective concentration of the chemical mutagenic agent is 1-6% (w / v), The method according to 2, more preferably characterized in that the duration of action is 0.5 to 2 hours.

4. Physical mutagenesis employs one or more combinations of ultraviolet radiation and ambient pressure room-temperature plasma, and preferably the duration of physical mutagenesis is 30 s to 90 s. Preferably, the output of the ambient pressure, room temperature plasma is 80 to 120 W, and the gas flow rate is 8 to 15 L / min; more preferably, the output of the ambient pressure, room temperature plasma is 100 W, and the gas flow rate is 10 L / min. More preferably, the method according to 2, characterized in that the output of ultraviolet radiation is 15 w to 20 watts and the irradiation distance is 20 to 30 cm.

5. In step (2), the bacterial cells are cultured statically in a culture medium. Preferably, the culture medium is YPD medium. More preferably, the pH of the culture medium is 5.0 to 6.

0. More preferably, the culture temperature is 28 to 32°C. More preferably, the method according to any one of claims 1 to 4, characterized in that the culture time is 30 to 60 minutes.

6. In step (3), the restored bacterial cells obtained in step (2) are cultured in an evolutionary medium containing an inhibitory substance, and the culture solution is collected and subinoculated into a new evolutionary medium and cultured, and this subinoculation is repeated 50 to 150 times. Preferably, the inhibitory substance is one or more of the following: 6-azauracil, 8-azaguanine, cycloheximide, 6-mercaptopurine, diaminopurine, 5-bromouracil, 5-fluorouracil. More preferably, the concentration of inhibitory substances in the culture medium after subculturing increases. More preferably, during the subculturing process, the concentration of the inhibitory substance in the culture medium increases by 50 to 1000 mg / L. More preferably, the culture is performed for 20-30 hours before subculturing, preferably for 24 hours. More preferably, the culture temperature is 28-33°C, and the culture is performed with shaking at 150-220 rpm. More preferably, the concentration of the inhibitory substance is 50 mg to 1000 mg / L, and more preferably, the concentration of the inhibitory substance is 50 mg to 500 mg / L. More preferably, the method according to any one of claims 1 to 5, characterized in that the initial concentration of the inhibitory substance is 50 mg to 100 mg / L.

7. In step (4), the mutagenesis library is stained with an RNA fluorescent dye, and strains with high fluorescence intensity are selected to obtain high RNA yeast. Preferably, the RNA fluorescent dye is one or more combinations of SYTO® RNASelect®, Hoechst 33258, Quant-iT® RiboGreen® RNA Reagent, or ethidium bromide, and preferably SYTO® RNASelect®. The method according to any one of claims 1 to 6.

8. The method according to any one of claims 1 to 7, further comprising the step (5) of scaling up verification.

9. The method according to step (5), characterized in that a fermentation verification is performed on the bacterial strain selected in step (4) in a fermenter and its RNA content is measured.

10. The fermentation medium, expressed by mass percentage, contains 2-6% glucose, 0.5-2% yeast extract, 0.2-0.4% magnesium sulfate heptahydrate, 0.2-0.4% zinc sulfate heptahydrate, 0.5-1.2% potassium dihydrogen phosphate, with the remainder being water. Preferably, the pH of the fermentation medium is 5.0 to 6.

0. More preferably, the fermentation stirring rotation speed is 300 to 600 rpm. More preferably, the method according to claim 9, characterized in that the fermentation aeration rate is 3 to 6 L / min.

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