Saccharomyces cerevisiae, fungicide, application and screening method

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to saccharomyces cerevisiae, a fungicide, application and a screening method. The invention provides a saccharomyces cerevisiae strain DXMC847, which is classified and named as saccharomyces cerevisiae, and is preserved in China Center for Type Culture Collection on November 18, 2024, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 20242592. The saccharomyces cerevisiae strain BY4741 is used as an original strain, and a method of combining ultraviolet mutagenesis with flow sorting is utilized to realize high-throughput screening of acid-resistant strains, so that the acid-resistant strains can be efficiently screened, and the yield of the acid-resistant strains is improved. And finally obtaining the acid-resistant saccharomyces cerevisiae DXMC847. The result of the embodiment shows that the content of pyruvic acid, L-malic acid and succinic acid in the fermentation liquor of the saccharomyces cerevisiae DXMC847 is obviously improved compared with that of the saccharomyces cerevisiae BY4741.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a brewer's yeast, a bacterial agent, and an application and screening method. Background Art

[0002] Saccharomyces cerevisiae Saccharomyces cerevisiae ) is the most commonly used biological strain in fermentation. It has the advantages of short growth cycle, strong fermentation ability and easy large-scale cultivation. At the same time, the culture medium obtained from the strain contains a variety of proteins, amino acids, vitamins, biologically active substances, etc., and is rich in nutrients. Therefore, brewer's yeast has always been the main object of basic and applied research.

[0003] In recent years, with the rapid development of synthetic biology, Saccharomyces cerevisiae has also been used to ferment and produce other metabolites with important industrial value, such as bioethanol, biobutanol, 2,3-butanediol, organic acids, resveratrol and farnesene.

[0004] Saccharomyces cerevisiae has the advantages of clear genetic background, strong genetic operability, and good fermentation performance. Since the birth of metabolic engineering, it has always been one of the preferred metabolic engineering chassis cells. With the continuous development of Saccharomyces cerevisiae metabolic engineering technology, the understanding of its metabolic network has been deepened, and the production efficiency of Saccharomyces cerevisiae as a platform cell factory has also been greatly improved. It has now been widely used in the large-scale production of various foods, chemical products and medicines. The ability to design cell resistance based on a specific environment may be used in basic research and biomanufacturing, and the acid resistance of Saccharomyces cerevisiae has always been of great concern to researchers. Acid stress is the most common abiotic stress encountered by yeast cells during the fermentation of acidic products such as organic acids, which seriously affects cell growth and metabolism. In order to alleviate the inhibitory effect of acid stress on cell growth and product synthesis, a large amount of neutralizers are often needed in the production process, which not only increases the cost and makes the downstream purification process more complicated, but also easily causes environmental pollution. Screening an acid-resistant Saccharomyces cerevisiae can solve this problem well. Summary of the invention

[0005] The purpose of the present invention is to provide a brewer's yeast, a bacterial agent, and an application and screening method. The brewer's yeast DXMC847 of the present invention has good acid resistance.

[0006] In order to solve the above technical problems, the following technical solutions are proposed: The present invention provides a strain of Saccharomyces cerevisiae ( Saccharomyces cerevisiae )DXMC847, classified as Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), deposited in the China Center for Type Culture Collection on November 18, 2024, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 20242592.

[0007] The present invention provides a bacterial agent, comprising the brewer's yeast DXMC847 described in the above technical solution.

[0008] The present invention provides the use of the brewer's yeast DXMC847 described in the above technical scheme or the bacterial agent described in the above technical scheme in preparing organic acids and / or increasing the yield of organic acids; the organic acids include short-chain organic acids, and the short-chain organic acids include one or more of L-malic acid, citric acid, succinic acid and pyruvic acid.

[0009] The present invention provides a method for preparing short-chain organic acids. The brewer's yeast DXMC847 or the bacterial agent described in the above technical solution is inoculated into a culture medium for fermentation culture to obtain a culture solution containing short-chain organic acids.

[0010] Preferably, the fermentation temperature is 28-32° C., and the fermentation time is 5-7 days.

[0011] The present invention provides a method for screening acid-resistant strains, comprising: The strain to be screened is inoculated into an acidic medium for cultivation and mutagenesis to obtain a mutagenesis solution; The stained mutagenesis solution is subjected to flow cytometry sorting, and the mutagenesis solution with higher fluorescence intensity is selected for single colony sorting to obtain multiple single colonies; The plurality of single colonies are subjected to multiple rounds of acid-resistant culture screening to obtain acid-resistant strains; The method of each round of acid-resistant culture screening is: inoculate the multiple single colonies in an acidic medium for cultivation, select OD 600 The taller colony; the number of rounds is ≥ 2 rounds.

[0012] Preferably, the acid stress source in the acidic medium includes one or more of L-malic acid, citric acid, lactic acid, succinic acid and pyruvic acid.

[0013] Preferably, the strain comprises Saccharomyces cerevisiae.

[0014] Preferably, the dye used in flow cytometry sorting includes SYTO-9.

[0015] Preferably, the flow cell sorting method comprises: washing, resuspending and diluting the mutagenesis solution or culture solution to obtain a bacterial suspension, and the OD of the bacterial suspension is 600 The volume ratio of bacterial suspension and dye for flow cytometry sorting is 1mL:3~5μL.

[0016] Beneficial effects of the present invention: The present invention provides a strain of brewer's yeast ( Saccharomyces cerevisiae)DXMC847, with a deposit number of CCTCC NO: M 20242592. The present invention uses the brewer's yeast strain BY4741 as the starting strain, and uses ultraviolet mutagenesis and cell flow sorting to achieve high-throughput screening of acid-resistant strains, and finally obtains acid-resistant brewer's yeast DXMC847. Brewer's yeast DXMC847 can grow and metabolize well in an environment of pH=2.3. The results of the embodiment show that the contents of pyruvic acid, L-malic acid and succinic acid in the fermentation broth of brewer's yeast DXMC847 are significantly increased compared with brewer's yeast BY4741. Brewer's yeast DXMC847 can be used as a potential chassis microorganism for fermenting short-chain organic acids, and is particularly suitable for the later transformation and production of pyruvic acid, L-malic acid and succinic acid. Brewer's yeast DXMC847 can grow and metabolize normally in a low-acid environment, providing guidance for the development of some organic acid products (especially pyruvic acid, L-malic acid and succinic acid).

[0017] The present invention provides a method for screening acid-resistant brewer's yeast. By combining ultraviolet mutagenesis with flow cytometry sorting, a suitable fluorescent dye is selected, a relationship between pH and fluorescence intensity system is established, and acid-resistant brewer's yeast is screened according to fluorescence intensity. A strain of acid-resistant brewer's yeast DXMC847 is screened by this method, and the brewer's yeast DXMC847 can grow and metabolize well in an environment with a pH of 2.3. This method has high precision and high speed when sorting a small number of cells. After being stained with fluorescent dye, the cells have fluorescence, which can be used as a marker on the surface of brewer's yeast. The cell surface has a specific marker of the marker, which can ensure the separation of specific cells, thereby screening suitable cells. The method of the present invention is convenient and fast, greatly shortens the screening time, and improves the screening efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the growth curve of BY4741 strain; Figure 2 This is a single cell scanning data analysis diagram of Saccharomyces cerevisiae in different culture media under different dye conditions in Example 2; Figure 3 This is a comparison of the fluorescence intensity of different samples in Example 3 in pH=2.5 and YPD medium; the left picture is pH=2.5 medium, and the right picture is YPD medium; Figure 4 This is a comparison chart of OD values ​​of colonies screened by flow cytometry under low-acid culture conditions in a 96-well plate; Figure 5 This is a comparison chart of OD values ​​of single colonies screened by flow cytometry in shaking tubes at different pH values; Figure 6 This is a comparison chart of OD values ​​of single colonies screened by flow cytometry in shake flasks at different pH values; Figure 7These are the results of the fermentation of BY4741 strain and DXMC847 to produce organic acids, where A is the liquid phase spectrum of the fermentation of BY4741 strain and DXMC847 to produce organic acids, and B, C, and D are the enlarged results of pyruvic acid, succinic acid, and L-malic acid in the fermentation broth, respectively.

[0019] Biological Deposit Description Saccharomyces cerevisiae Saccharomyces cerevisiae )DXMC847, classified as Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), deposited in the China Center for Type Culture Collection on November 18, 2024, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 20242592. DETAILED DESCRIPTION

[0020] The present invention provides a strain of Saccharomyces cerevisiae DXMC847, with a deposit number of CCTCC NO: M 20242592. The ITS gene sequence of the Saccharomyces cerevisiae DXMC847 of the present invention is shown in SEQ ID NO.1.

[0021] SEQ ID NO.1:。

[0022] The brewer's yeast of the present invention uses the brewer's yeast BY4741 as a starting strain, and uses ultraviolet mutagenesis combined with a flow sorting method to achieve high-throughput screening of acid-resistant strains to obtain acid-resistant brewer's yeast DXMC847. The brewer's yeast DXMC847 grows well in an environment with a pH of 2.3. The results of the embodiment show that the yields of pyruvic acid, L-malic acid and succinic acid in the fermentation broth of the brewer's yeast DXMC847 are significantly increased compared with the brewer's yeast BY4741. The brewer's yeast DXMC847 obtained by screening can be used as a potential chassis microorganism for fermenting short-chain organic acids, and the organic acids are L-malic acid, citric acid, succinic acid and pyruvic acid, and is particularly suitable for the production of succinic acid, L-malic acid and pyruvic acid.

[0023] The present invention provides a bacterial agent, comprising the brewer's yeast DXMC847 described in the above technical solution.

[0024] As an optional embodiment, the application form of the Saccharomyces cerevisiae DXMC847 of the present invention includes seed culture solution.

[0025] As an optional embodiment, the method for preparing the culture solution of the present invention comprises the following steps: inoculating the brewer's yeast DXMC847 into a culture medium for culturing to obtain a culture solution. As another optional embodiment, the present invention inoculates a single colony of the brewer's yeast DXMC847 into a culture medium for seed culture to obtain a seed culture solution. The present invention has no special limitation on the inoculation method, and a conventional method may be used. As an optional embodiment, the temperature of the seed culture of the present invention is 28°C to 32°C, or may be 30°C; in a specific embodiment of the present invention, the culture temperature is 28, 29, 30, 31 or 32°C. The time of the seed culture of the present invention is 10 to 15h. In a specific embodiment of the present invention, the time of the seed culture is 10, 11, 12, 13, 14 or 15h. The culture medium used for the culture of the present invention includes YPD liquid culture medium. As an optional embodiment, the OD of the seed culture solution is 600 2~8.

[0026] The present invention provides the use of the saccharomyces cerevisiae DXMC847 or the bacterial agent described in the above technical solution in preparing short-chain organic acids and / or increasing the yield of short-chain organic acids; the short-chain organic acids of the present invention include one or more of L-malic acid, citric acid, succinic acid and pyruvic acid, preferably succinic acid, L-malic acid and pyruvic acid. The results of the embodiment show that the yield of pyruvic acid, L-malic acid and succinic acid in the fermentation broth of saccharomyces cerevisiae DXMC847 is significantly increased compared with that of saccharomyces cerevisiae BY4741.

[0027] The present invention provides a method for preparing short-chain organic acids, comprising the following steps: inoculating the brewer's yeast DXMC847 described in the above technical solution or the bacterial agent described in the above technical solution into a culture medium for fermentation culture to obtain a culture solution, wherein the culture solution contains short-chain organic acids.

[0028] As an optional embodiment, when the application form of the cerevisiae yeast DXMC847 is a single colony, the present invention does not specifically limit the method of inoculation, and a conventional method can be used. When the application form of the cerevisiae yeast DXMC847 is a seed culture solution, the inoculation amount of the seed culture solution is 2% to 5% of the volume of the culture medium, or it can be 3% to 4%. In a specific embodiment of the present invention, the inoculation amount is 2%, 3%, 4% or 5% of the volume of the culture medium.

[0029] As an optional embodiment, the composition of the culture medium of the present invention includes: 50g / L glucose, 15g / L yeast powder, 12g / L (NH4)2SO4, 5g / L K2HPO4, 0.1g / L CaCl2·H2O, 0.5g / L ZnSO4·7H2O, 6g / L MgSO4·7H2O. In the fermentation and culture process of the present invention, glucose batch feeding is performed. As an optional embodiment, the method of glucose batch feeding includes: when the glucose content in the fermentation and culture system is less than 20 g / L, adding a glucose mother solution with a mass concentration of 80% to control the glucose content in the fermentation and culture to 20-30 g / L. In the fermentation and culture process of the present invention, samples are taken at regular intervals to detect the residual sugar content of glucose, and the time of glucose batch feeding is determined according to the consumption of glucose.

[0030] As an optional embodiment, the temperature of the fermentation culture is 28-32°C, or 30°C. The fermentation culture time is 5-7 days, or 6 days; the fermentation culture speed is 200-240r / min, or 220r / min. The temperature, culture time, and speed of the present invention are set to increase the yield of L-malic acid, succinic acid, pyruvic acid, and citric acid of Saccharomyces cerevisiae DXMC847.

[0031] The present invention provides a method for screening acid-resistant strains, comprising: The strain to be screened is inoculated into an acidic medium for cultivation and mutagenesis to obtain a mutagenesis solution; The stained mutagenesis solution is subjected to flow cytometry sorting, and the mutagenesis solution with higher fluorescence intensity is selected for single colony sorting to obtain multiple single colonies; A plurality of single colonies are subjected to multiple rounds of acid-resistant culture screening to obtain acid-resistant strains; The method of each round of acid-resistant culture screening is: inoculate the multiple single colonies in an acidic medium for cultivation, select OD 600 Higher strains; the number of rounds is ≥ 2 rounds.

[0032] The present invention can adjust the pH value of the acidic culture medium according to the growth conditions of the bacterial colony.

[0033] The present invention cultivates and mutates the strain to be screened in an acidic medium to obtain a mutagenic solution. As an optional embodiment, the strain includes Saccharomyces cerevisiae. In a specific embodiment of the present invention, Saccharomyces cerevisiae is screened by the screening method of the present invention to obtain acid-resistant Saccharomyces cerevisiae DXMC847. As an optional embodiment, the acid stress pressure source in the acidic medium of the present invention includes one or more of L-malic acid, citric acid, lactic acid, succinic acid, and pyruvic acid.

[0034] As an optional embodiment, the mutagenesis of the present invention includes ultraviolet mutagenesis. As an optional embodiment, the present invention performs ultraviolet mutagenesis on the culture solution cultured to the logarithmic phase; as an optional embodiment, the time of ultraviolet mutagenesis of the present invention is 100~150s. As an optional embodiment, the present invention records the ultraviolet mutagenesis and culture as one round of mutagenesis, and the present invention preferably performs greater than or equal to 4 rounds of mutagenesis. After completing one round of mutagenesis, the present invention obtains a mutagenesis solution, and the mutagenesis solution is inoculated into the culture medium as an inoculum for the next round of mutagenesis. As an optional embodiment, when performing the next round of mutagenesis, the inoculum amount of the mutagenesis solution of the present invention is 0.5‰~2% of the volume of the culture medium.

[0035] After all the mutagenesis is completed, a suitable fluorescent dye is selected to dye the above-mentioned mutagenic solution. Fluorescence detection is performed on the stained mutagenic sample by flow cytometry, and a mutagenic solution with stronger fluorescence intensity is selected for flow cytometry single colony sorting. In a specific embodiment of the present invention, after nine rounds of mutagenesis, the obtained mutagenic solution is dyed for fluorescence detection, and the fluorescence intensity of the mutagenic solution obtained by the fourth, seventh and eighth rounds of mutagenesis is stronger, and the mutagenic solution obtained by the fourth, seventh and eighth rounds of mutagenesis is subjected to flow cytometry sorting. The present invention performs flow cytometry single colony sorting on the mutagenic solution for ≥1 times. In a specific embodiment of the present invention, only one flow cytometry sorting is performed on the mutagenic solution, and multiple flow cytometry single colony sortings can also be performed. The present invention only performs flow cytometry single colony sorting, and a mutagenic solution with higher fluorescence intensity can also be selected for colony collection, and single colony sorting is performed on the colony again until a strain with better acid resistance is obtained.

[0036] As an optional embodiment, the dye used for flow cytometry sorting of the present invention includes SYTO-9. SYTO-9 is used as a dye because the fluorescence difference of the dye is large when detecting culture fluids with different pH values, and the difference is significant during sorting, making it easier to sort out acid-resistant strains.

[0037] As an optional embodiment, the flow cell sorting method of the present invention comprises: washing, resuspending and diluting the mutagenesis solution or culture solution to obtain a bacterial suspension, and the OD of the bacterial suspension is 600 is 0.8-1.5, the bacterial suspension is stained with a dye, the volume ratio of the bacterial suspension to the dye is 1 mL: 3-5 μL, a flow cytometer is used for fluorescence detection and sorting, and single colonies are sorted for samples with high fluorescence intensity. As an optional embodiment, the washing and resuspension of the present invention are completed using physiological saline.

[0038] The method of each round of acid-resistant culture screening of the present invention is: the plurality of single bacterial colonies are inoculated into an acidic medium for culture, and the OD 600 As another optional embodiment, the present invention performs multiple rounds of acid-resistant culture screening on the multiple single colonies, inoculates the single colonies obtained by the sorting into an acidic culture medium for a first culture, screens acid-resistant single colonies based on whether colonies are formed, and screens out second acid-resistant single colonies; performs a second culture on the second acid-resistant single colonies, and further performs a second culture on the obtained culture fluid based on OD 600 Acid-resistant strains are screened to obtain a third acid-resistant single colony; the present invention can gradually eliminate and screen according to the base size of the single colonies obtained by sorting until the strain with the best growth state of the strain is selected. The present invention performs a total of four rounds of screening processes, and the pH value of the culture solution is reduced during the third round of screening. The present invention does not specifically limit the parameters of the flow cell sorting, and conventional methods can be used. In a specific embodiment of the present invention, multiple single colonies obtained by flow sorting are subjected to four rounds of culture sorting to obtain acid-resistant brewer's yeast DXMC847.

[0039] The present invention provides a method for high-throughput screening of acid-resistant brewer's yeast. The method can quickly screen acid-resistant brewer's yeast in a short time, and can also screen strains of other genera, not limited to the strain of brewer's yeast.

[0040] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0041] The composition of YPD solid culture medium is: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract and 20 g / L agar.

[0042] The composition of YPD liquid culture medium is: 20 g / L glucose, 20 g / L peptone and 10 g / L yeast extract.

[0043] Medium 1 is a YPD liquid medium with a pH of 6.5. Medium 2 is a YPD liquid medium with a pH of 2.5, which is an acidic medium and the pH is adjusted using L-malic acid. Medium 3 is a YPD liquid medium with a pH of 2.3 and the pH is adjusted using L-malic acid.

[0044] Example 1 A method for high-throughput screening of acid-tolerant Saccharomyces cerevisiae, wherein the selection of acidic culture medium and the ultraviolet mutagenesis screening method are as follows: 1. Take the brewer's yeast BY4741 preserved in a glycerol tube, draw three lines on the YPD solid medium plate, and obtain a single colony after cultivation. Pick a single colony and inoculate it in a 250mL shake flask, where the liquid volume of YPD liquid medium is 30~50mL, and activate the strain. The inoculated shake flask is placed on a shaker and shaken at 30℃ and 220r / min. During the cultivation process, sterile sampling is performed every 2h.

[0045] All samples were tested by UV spectrophotometer at an absorbance of 600 nm. 600 The value was in the range of 0.2 to 1, and the UV spectrophotometer was zeroed with uninoculated YPD liquid culture medium during the test.

[0046] Until the OD of the sample is 600 When the value tends to be stable or shows a downward trend, stop sampling. With the sampling time as the horizontal axis, the actual optical density value (i.e. OD 600 Value) as the vertical axis, draw the growth curve, the growth curve is shown in Figure 1 ,according to Figure 1 It can be seen that the bacterial solution in the logarithmic phase was obtained after culturing for 10 to 16 hours. The OD of the bacterial solution in the logarithmic phase was 600 2~8.

[0047] 2. The activated Saccharomyces cerevisiae BY4741 was inoculated into acidic culture media with different pH values ​​(pH was adjusted using L-malic acid) at different inoculation rates, and the growth of Saccharomyces cerevisiae BY4741 in acidic culture media with different pH values ​​was observed. Saccharomyces cerevisiae BY4741 grew very slowly in a culture medium with pH = 2.5 and did not grow in a culture medium with pH = 2.3.

[0048] 3. Ultraviolet mutagenesis: Saccharomyces cerevisiae BY4741 was cultured in YPD liquid medium for 14 hours to obtain a culture solution in the logarithmic phase; the culture solution in the logarithmic phase was centrifuged and the supernatant was discarded to obtain the bacteria. The bacteria were gradient diluted with physiological saline to obtain dilutions of different dilution multiples. 100 μL of the dilutions of different dilution multiples were spread on YPD solid medium plates, inverted and cultured at 30°C, and the dilution gradient was determined when the number of colonies was about 100-300 for subsequent experiments. The determined dilution gradient was 10 -4 .

[0049] Turn on the UV lamp to preheat for 30 minutes in advance, and take the dilution gradient of 10 -4 3mL of bacterial solution was placed in a 60mm diameter culture dish for UV treatment for 0s, 30s, 60s, 90s, 120s and 150s, and 3 parallel experiments were set for each treatment time. 100μL of the mutagenic bacterial solution was evenly spread on a YPD solid culture medium plate, inverted and cultured at 30℃ for about 24h, and the colonies were counted. The UV treatment time with a lethality of about 90% was used as the subsequent UV mutagenesis treatment time, and the UV mutagenesis time was determined to be 120s.

[0050] Example 2 A method for high-throughput screening of acid-resistant cerevisiae yeast, wherein the fluorescent dye screening method is as follows: Propidium iodide (PI) is a commonly used red fluorescent nuclear and chromosome counterstain. Since propidium iodide cannot penetrate the membrane into living cells and can penetrate damaged cells, it is often used to identify cell viability and detect dead cells in a population. PI dye is suitable for fluorescence microscopy and flow cytometry. SYTO-9 is a green fluorescent nucleic acid dye that can penetrate the cell membrane and can be used to stain RNA and DNA in living cells, dead cells, and bacteria.

[0051] 1. Saccharomyces cerevisiae (BY4741) was inoculated into medium 1 and medium 2 respectively, and then cultured. The bacterial concentration was detected under the condition of ultraviolet absorbance at 600nm.

[0052] 2. Wash, resuspend and dilute the samples obtained from culture medium 1 and culture medium 2 with physiological saline to obtain bacterial suspension. The OD of bacterial suspension is 600 The bacterial suspension is dyed at a volume ratio of 1 mL:3 μL of the bacterial suspension and the dye, and the dyes are PI dye, SYTO-9 dye and mixed dye, and the volume ratio of PI dye and SYTO-9 dye in the mixed dye is 1:1.

[0053] 3. After staining, all samples were scanned for fluorescence spectrum at excitation light of 470nm and emission light of 513nm~850nm. Figure 2 .

[0054] Figure 2 The blue line in the figure represents medium 1, i.e., YPD, and the orange line represents medium 2, i.e., YPD medium with a pH of 2.5. Select appropriate fluorescent dyes for bacterial staining based on the scanning results.

[0055] According to the attached Figure 2 The results show that when a single cell is stained with PI dye: PI dye is used to identify dead cells in a population. The fluorescence intensity of cells cultured in pH=2.5 acidic medium (i.e., medium 2) is greater than that of cells cultured in medium 1, indicating that the lower the pH, the more dead cells there are. The maximum fluorescence value of this dye is 635nm wavelength.

[0056] When single cells were stained with SYTO-9 dye: the fluorescence intensity of cells cultured in medium 1 was greater than that of cells cultured in medium 2, indicating that the lower the pH, the lower the cell viability. The maximum fluorescence value of this dye was 505 nm wavelength.

[0057] When a single cell is stained with a mixed dye: with 615nm as the boundary, the fluorescence intensity of the strain grown in culture medium 1 is greater when the wavelength is less than 615nm; the fluorescence intensity of the strain grown in culture medium 2 is greater than 615nm, indicating that the lower the pH, the more dead cells there are and the lower the cell viability.

[0058] In order to further select the staining method with greater fluorescence difference, a blank control group of normal saline was set up, and the staining experiments of medium 1 and medium 2 were repeated. At the same time, the normal saline was tested as follows: Medium 1 and medium 2 were stained and the bacterial concentration was detected according to steps 1 to 3 above.

[0059] Normal saline group: Staining and bacterial concentration detection were performed according to the above steps 1 to 3, except that the bacterial suspension in step 2 was replaced with normal saline, and staining was performed at a volume ratio of 1 mL:3 μL of normal saline to dye.

[0060] After staining, each group used a fluorescence detection microplate reader to detect single-point fluorescence values ​​under the conditions of excitation wavelength 470nm and emission wavelength 505nm. The results are shown in Table 1. It can be seen that the fluorescence values ​​under different pH values ​​and different dye conditions have different differences. The relationship between pH and fluorescence intensity can be established. According to the difference results, SYTO-9 fluorescent dye with significant error is preferred for staining.

[0061] Table 1 Analysis of single cell fluorescence values ​​of Saccharomyces cerevisiae in different culture media under different dye conditions

[0062] Example 3 A method for high-throughput screening of acid-tolerant Saccharomyces cerevisiae, wherein the flow sorting method is as follows: 1. Cell culture Treatment group 1: Saccharomyces cerevisiae (BY4741) was inoculated into culture medium 1 and cultured at 30°C and 220 r / min for 12 h to obtain the first generation culture solution (referred to as culture solution 1 as a control).

[0063] First round of mutagenesis: The culture medium 1 was subjected to the first UV mutagenesis for 120 seconds. Then, the culture medium after mutagenesis was transferred to the culture medium 1 at an inoculation amount of 1‰ of the culture medium volume and cultured at 30°C and 220r / min for 18 hours to obtain the mutant 1 generation culture medium (recorded as culture medium 2).

[0064] Second round of mutagenesis: The culture medium of the first generation of mutation was subjected to a second UV mutagenesis, and the UV mutagenesis time was 120s. After that, the culture medium after mutagenesis was transferred to the culture medium 1 at an inoculation amount of 1‰ of the culture medium volume and cultured at 30°C and 220r / min for 20h to obtain the culture medium of the second generation of mutation (recorded as culture medium 3).

[0065] The third round of mutagenesis: The second generation culture fluid was subjected to the third UV mutagenesis, and the UV mutagenesis time was 120 seconds. After that, the culture fluid after mutagenesis was transferred to the culture medium 1 at an inoculation amount of 1‰ of the culture medium volume and cultured at 30°C and 220r / min for 25 hours to obtain the third generation culture fluid of mutation (recorded as culture fluid 4).

[0066] According to the method of each round of mutagenesis culture mentioned above, the fourth round of mutagenesis, the fifth round of mutagenesis, the sixth round of mutagenesis, the seventh round of mutagenesis, the eighth round of mutagenesis, and the ninth round of mutagenesis were carried out to obtain the 4th generation mutation culture fluid (recorded as culture fluid 5, 4th generation (mutation)), the 5th generation mutation culture fluid (recorded as culture fluid 6, 5th generation (mutation)), the 6th generation mutation culture fluid (recorded as culture fluid 7, 6th generation (mutation)), the 7th generation mutation culture fluid (recorded as culture fluid 8), the 8th generation mutation culture fluid (recorded as culture fluid 9) and the 9th generation mutation culture fluid (recorded as culture fluid 10). The determination of the culture time for each round is based on the fact that the liquid becomes obviously turbid.

[0067] Treatment group 2: Saccharomyces cerevisiae (BY4741) was inoculated in medium 2 and cultured at 30°C and 220 r / min for 20 h; the first generation culture fluid of medium 2 was obtained (recorded as culture fluid 1-2.5, as a control); First round of mutagenesis: The first generation culture medium of medium 2 was subjected to the first UV mutagenesis for 120 seconds. Then, the culture medium after mutagenesis was transferred to medium 2 at an inoculation amount of 1‰ of the volume of the culture medium and cultured at 30°C and 220r / min for 30 hours to obtain mutant 1-1 culture medium (recorded as culture medium 2-2.5); Second round of mutagenesis: The culture medium of mutant 1-1 was subjected to a second UV mutagenesis for 120 seconds. Then, the culture medium after mutagenesis was transferred to medium 2 at an inoculation volume of 5‰ of the volume of the culture medium and cultured at 30°C and 220r / min for 33h to obtain the culture medium of mutant 2-1 (recorded as culture medium 3-2.5).

[0068] The third round of mutagenesis: The mutant 2-1 culture solution was subjected to the third UV mutagenesis for 120 seconds. The culture solution after mutagenesis was then transferred to the culture medium 2 at an inoculation volume of 1% of the culture medium volume and cultured at 30°C and 220 r / min for 40 hours to obtain the mutant 3-1 culture solution (recorded as culture solution 4-2.5).

[0069] According to the method of each round of mutagenesis culture mentioned above, the fourth round of mutagenesis, the fifth round of mutagenesis, the sixth round of mutagenesis, the seventh round of mutagenesis, the eighth round of mutagenesis and the ninth round of mutagenesis were carried out to obtain mutation 4-1 culture solution (recorded as culture solution 5-2.5, 4th generation (mutation)), mutation 5-1 culture solution (recorded as culture solution 6-2.5, 5th generation (mutation)), mutation 6-1 culture solution (recorded as culture solution 7-2.5, 6th generation (mutation)), mutation 7-1 culture solution (recorded as culture solution 8-2.5, 7th generation (mutation)), mutation 8-1 culture solution (recorded as culture solution 9-2.5, 8th generation (mutation)) and mutation 9-1 culture solution (recorded as culture solution 10-2.5, 9th generation (mutation)), respectively. The inoculation amount of each round of mutagenesis was based on the growth capacity of the cells, and the culture time was based on the turbidity of the cells.

[0070] The mutant culture fluid obtained in each round of cultivation was sampled.

[0071] 2. Screening of acid-resistant strains by flow cytometry Take 1 mL of culture medium 1, culture medium of mutation 4 to culture medium of mutation 9, culture medium 1-2.5, culture medium of mutation 4-1 to culture medium of mutation 9-1, wash the cells in the culture medium, resuspend them in physiological saline and dilute to OD 600=1.0, and the staining operation was performed according to the SYTO-9 dye staining method of Example 2. After that, each sample was loaded onto the machine, and samples with higher fluorescence intensity were selected according to the fluorescence intensity trend. The ultraviolet mutant strains with stronger green fluorescence were sorted using a flow cytometer. The specific sorting conditions were: excitation light at 488nm, fluorescence detection at 535nm, sample pressure at 60psi, and nozzle diameter set to 70μm. The obtained data were analyzed using Beckman Summit 5.2 software for strain grouping. The sample flow fluorescence detection results are shown in Figure 3 (The left picture is pH=2.5 culture medium; the right picture is YPD culture medium, i.e. culture medium 1). It can be seen that after all the samples were put into the machine, the strains were continuously domesticated by ultraviolet mutagenesis, the fluorescence intensity was continuously enhanced, the strain activity was continuously improved, and the acid resistance of the strains was continuously improved. The fluorescence domestication range of the strains cultured in low-acid culture medium was wider and the domestication effect was better. Among them, the fluorescence signals of mutation 4-1 culture medium, mutation 7-1 culture medium and mutation 8-1 culture medium were stronger.

[0072] According to the data analysis results in Beckman Summit 5.2 software, a gate of 0.01% of the total cells was set to collect cells with higher fluorescence intensity. Specifically: Comprehensively evaluate and select the strain samples of the first-generation culture fluid (control fluid), mutation 4-1 culture fluid, mutation 7-1 culture fluid, and mutation 8-1 culture fluid obtained in culture medium 2 for flow cytometer sorting. Flow cytometer sorting will use electrical stimulation to turn cells into single cells through the sorting channel. The collected cells are all single cells. A total of 590 single colonies were sorted, of which 60 single colonies were sorted in the control fluid (single colonies were placed in 1 96-well plate, that is, the number of sorting plates was 1 96-well plate), and 120 single bacteria were sorted in the mutation 4-1 culture fluid. 170 single colonies were sorted in mutation 7-1 culture medium (single colonies were placed in 3 96-well plates, i.e., the number of sorting plates was 3 96-well plates), 240 single colonies were sorted in mutation 8-1 culture medium (single colonies were placed in 4 96-well plates, i.e., the number of sorting plates was 4 96-well plates), 590 single colonies were cultured in 96-well plates containing culture medium 2 at 30°C and 220r / min, and the grown single colonies were selected for subsequent screening of acid-resistant single strains.

[0073] Example 4 A method for high-throughput screening of acid-resistant brewer's yeast, wherein the sorted samples are further subjected to an acid-resistant screening method, and the results are as follows: The growth status of the single colonies screened in the 96-well plate in Example 3 was regularly observed. A total of 79 single colonies were grown (strain numbers 1 to 79), specifically: 5 single colonies were grown in the control solution (strain numbers 1 to 5), 17 single colonies were grown in the mutant 4-1 culture solution (strain numbers 6 to 22), 31 single colonies were grown in the mutant 7-1 culture solution (strain numbers 23 to 53), and 26 single colonies were grown in the mutant 8-1 culture solution (strain numbers 54 to 79).

[0074] Afterwards, the growing strain was transferred to a 96-well plate according to a volume ratio of 1% inoculation amount. The 96-well plate contained culture medium 2. Two parallel plate experiments were set up. The two parallel plate experiments were named 2.5-1 and 2.5-2, respectively. Both were shaken and cultured at 30°C and 220r / min, and the growth status of the strain was continuously observed.

[0075] When the culture medium becomes turbid, the plate is scanned with an ELISA reader at a scanning wavelength of 600 nm, and acid-resistant strains are further screened based on the detected values ​​(see Figure 4 ). According to OD 600 Thirteen relatively acid-resistant strains were further screened, namely strain No. 11, strain No. 12, strain No. 13, strain No. 21, strain No. 29, strain No. 35, strain No. 45, strain No. 72, strain No. 73, strain No. 74, strain No. 77, strain No. 78 and strain No. 79.

[0076] The 13 strains were renamed according to the sorting samples, the number of sorting plates and the number of grown colonies, namely, 4th generation-1-6 (strain numbered 11), 4th generation-1-7 (strain numbered 12), 4th generation-1-8 (strain numbered 13), 4th generation-1-16 (strain numbered 21), 7th generation-1-7 (strain numbered 29), 7th generation-2-3 (strain numbered 35), 7th generation-3-1 (strain numbered 45), 8th generation-4-1 (strain numbered 72), 8th generation-4-2 (strain numbered 73), 8th generation-4-3 (strain numbered 74), 8th generation-4-6 (strain numbered 77), 8th generation-4-7 (strain numbered 78) and 8th generation-4-8 (strain numbered 79).

[0077] The second strain (numbered as strain 2) from the 13 acid-resistant strains and the control liquid was transferred to a 10mL sterile shake tube (3mL of liquid) at a volume ratio of 2%. The pH of the culture medium in the shake tube was adjusted to 2.3 (i.e., culture medium 3) with L-malic acid, and cultured at 30°C and 220r / min under shaking conditions, and the growth status of the strain was continuously observed.

[0078] When the culture medium becomes turbid, the plate is scanned with an ELISA reader at a scanning wavelength of 600 nm, and acid-resistant strains are further screened based on the detected values ​​(see Figure 5 ). Three strains were further screened, namely 7th generation-1-7, 8th generation-4-2 and 8th generation-4-7, and the second strain (numbered as strain 2) was selected from the control solution for further comparative verification. The three acid-resistant strains screened were transferred to a 250mL shake flask (liquid volume 30mL) at an inoculation volume of 2%. The pH of the culture medium in the 250mL shake flask was adjusted to 2.3 with L-malic acid (culture medium 3), and the culture was shaken at 30℃ and 220r / min, and the growth status of the strain was continuously observed (see Figure 6 ), the 8th generation-4-7 strain was recorded as DXMC847 as the screened acid-resistant strain, and the growth performance of the strain obtained by screening was significantly better than that of the control strain.

[0079] Example 5 The acid-resistant Saccharomyces cerevisiae strains DXMC847 and BY4741 obtained in Example 4 were fermented in 250 mL shake flasks, and the types and contents of organic acids in the fermentation supernatants were detected by high performance liquid chromatography (HPLC). The required culture medium, culture conditions and detection methods are as follows: Preparation of seed culture solution: BY4741 strain and DXMC847 strain were prepared into glycerol tube strains for preservation, and the preserved strains were inoculated into YPD solid culture medium for activation. Single colonies of the activated strains were selected and inoculated into 15mL YPD liquid shake tubes for seed solution preparation (the medium volume of the shake tube was 3mL), and then cultured at 30℃ and 220r / min for 14h until the logarithmic growth phase to obtain BY4741 seed solution and DXMC847 seed solution.

[0080] Shake flask fermentation culture: BY4741 seed solution and DXMC847 seed solution were inoculated at a volume ratio of 3%, respectively. Specifically, 1.5 mL of seed solution was inoculated into a baffled shake flask containing fermentation medium. The volume of the shake flask was 250 mL, and the liquid volume was 50 mL. Fermentation culture was carried out at 30°C and 220 r / min. The fermentation liquid obtained after 144 h of fermentation culture was centrifuged at 8000 r / min for 2 min. The supernatant of the fermentation liquid was passed through a 0.22 μm aqueous membrane to prepare BY4741 liquid phase detection samples and DXMC847 liquid phase detection samples.

[0081] The composition of the fermentation medium was: glucose 50 g / L, yeast powder 15 g / L, (NH4)2SO4 12 g / L, K2HPO4 5 g / L, CaCl2·H2O 0.1 g / L, ZnSO4·7H2O 0.5 g / L, MgSO4·7H2O 6 g / L. During the fermentation process, glucose was supplemented by batch feeding, specifically: sampling was carried out at regular intervals to detect residual sugar. When the glucose concentration was lower than 20 g / L, 80% glucose mother liquor was added according to the sugar consumption to control the glucose content in the fermentation broth to 20-30 g / L.

[0082] Detection method of organic acids in fermentation broth: chromatographic column Agilent ZORBAX Eclipse XDB-C18 (5μL, 4.6nm*250nm), mobile phase: 0.1mol / L KH2PO4 solution, the pH of KH2PO4 solution was adjusted to 2.8 with phosphoric acid, flow rate 0.5mL / min, detection wavelength 210nm, injection volume 20μL, column temperature 20℃.

[0083] The liquid phase test results of BY4741 and DXMC847 are as follows Figure 7 As shown in A, the blue line is the organic acid detection result in the fermentation broth of BY4741 strain, and the orange line is the organic acid detection result in the fermentation broth of DXMC847 strain. Figure 7 Middle B, Figure 7 Middle C, Figure 7 D in the middle are the corresponding results of pyruvate, succinate and L-malate in the fermentation broth. It can be seen that L-malate, succinate, pyruvate and citric acid can be detected in the fermentation broth of BY4741 strain and DXMC847 strain. According to the data in Table 2, it can be seen that the original chassis cell BY4741 strain has a very weak ability to produce short-chain organic acids. The DXMC847 strain obtained by high-throughput screening not only has good acid resistance, but also has a significant improvement in its acid production ability. Among them, the content of pyruvate, L-malate and succinate in the fermentation broth of DXMC847 strain is greatly improved compared with the concentration of the original strain BY4741. It can be seen that the DXMC847 strain can be used as a potential chassis microorganism for fermenting short-chain organic acids, especially suitable for the later transformation and production of pyruvate, L-malate and succinate.

[0084] Table 2 Organic acid content in fermentation broth of BY4741 strain and DXMC847 strain

[0085] Example 6 The strain DXMC847 was prepared into a glycerol tube strain for preservation, and the preserved strain was resuscitated and inoculated into an acidic YPD medium with a pH of 2.3 (the pH was adjusted with L-malic acid, citric acid, lactic acid, succinic acid, and pyruvic acid, respectively). After 5 to 10 rounds of continuous subculture, the DXMC847 strain could grow in the acidic medium and had good stability.

[0086] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Saccharomyces cerevisiae ( Saccharomyces cerevisiae )DXMC847, classified as Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), deposited in the China Center for Type Culture Collection on November 18, 2024, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 20242592.

2. A bacterial agent, characterized in that The invention comprises the Saccharomyces cerevisiae DXMC847 as claimed in claim 1.

3. Use of the brewer's yeast DXMC847 according to claim 1 or the bacterial agent according to claim 2 in preparing organic acids and / or increasing the yield of organic acids; the organic acids include short-chain organic acids, and the short-chain organic acids include one or more of L-malic acid, citric acid, succinic acid and pyruvic acid.

4. A method for preparing a short-chain organic acid, characterized in that: The brewer's yeast DXMC847 according to claim 1 or the bacterial agent according to claim 2 is inoculated into a culture medium for fermentation culture to obtain a culture solution containing short-chain organic acids.

5. The preparation method according to claim 4, characterized in that: The fermentation temperature is 28-32° C., and the fermentation time is 5-7 days.

6. A method for screening acid-resistant strains, characterized in that: include: The strain to be screened is inoculated into an acidic medium for cultivation and mutagenesis to obtain a mutagenesis solution; The stained mutagenesis solution is subjected to flow cytometry sorting, and the mutagenesis solution with higher fluorescence intensity is selected for single colony sorting to obtain multiple single colonies; The plurality of single colonies are subjected to multiple rounds of acid-resistant culture screening to obtain acid-resistant strains; The method of each round of acid-resistant culture screening is: inoculate the multiple single colonies in an acidic medium for cultivation, select OD 600 The taller colony; the number of rounds is ≥ 2 rounds.

7. The screening method according to claim 6, characterized in that The acid stress source in the acidic medium includes one or more of L-malic acid, citric acid, lactic acid, succinic acid and pyruvic acid.

8. The screening method according to claim 6, characterized in that The strains include Saccharomyces cerevisiae.

9. The screening method according to claim 6, characterized in that The dye used in the flow cytometry sorting includes SYTO-9.

10. The screening method according to claim 6 or 9, characterized in that The flow cell sorting method comprises: washing, resuspending and diluting the mutagenesis solution or culture solution to obtain a bacterial suspension; the OD value of the bacterial suspension is 600 The volume ratio of bacterial suspension and dye for flow cytometry sorting is 1mL:3~5μL.

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