High-nucleic acid Kluyveromyces marxianus mutant strain and its application
Through screening and mutagenesis breeding, the Max Kluvia strain CJA0022 with high nucleic acid content was obtained, which solved the problem of low RNA content in wild-type yeasts, achieved efficient RNA production, reduced production costs, and enhanced its application value in the fields of food, medicine and agriculture.
Patent Information
- Application Number
- CN202510031200.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-09
- Publication Date
- 2025-08-19
- Estimated Expiration
- 2045-01-09
AI Technical Summary
The existing Max Kluvier yeast has a low RNA content, which leads to a higher cost of producing RNA and nucleotide products, affecting its competitiveness in the food and medicine fields.
Through chemical mutagenesis, physical mutagenesis and genetic stability verification, the Max Kluvia strain CJA0022 with high nucleic acid content was screened, and the nucleic acid content in single cells can reach 15% and can be stable in inheritance.
It has increased the nucleic acid content and growth rate of the Max Kluvia strain, reduced production costs, and expanded its application potential in the fields of food, medicine and agriculture.
Smart Images
Figure CN119410505B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology and specifically relates to a high nucleic acid Kluyveromyces marxianus ( Kluyveromyces marxianus ) mutant strains and their applications. Background Art
[0002] Ribonucleic acid (RNA), a type of nucleic acid, plays an important role in gene transcription and protein biosynthesis. At the same time, RNA and its degradation products are also widely used in food, medicine and agriculture. The current method of producing RNA is mainly from Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) and Candida ( Candida ), but Candida is not a food-safe yeast and Saccharomyces cerevisiae cannot naturally utilize lactose. Kluyveromyces marxianus ) is an unconventional yeast with the characteristics of fast growth rate and wide metabolic substrates. It is also a biosafety (GRAS) food-grade microorganism and can be used for fermentation in the food industry and production of drugs.
[0003] Due to the low RNA content of wild K. marxianus yeast, the cost of RNA, nucleotides, and other products derived from it is relatively high. Therefore, it is necessary to screen for a strain of K. marxianus yeast with a high nucleic acid content to reduce production costs and improve product competitiveness. Summary of the Invention
[0004] In order to overcome the above problems, the purpose of the present application is to provide a new strain of Kluyveromyces marxianus with high biomass and nucleic acid content.
[0005] In one aspect, the present disclosure provides a high nucleic acid content Kluyveromyces marxianus, which is classified as Kluyveromyces marxianus ( Kluyveromyces marxianus ) CJA0022, its deposit number is CCTCC No. M20242475.
[0006] The Kluyveromyces marxianus strain described in the present disclosure has been deposited in the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China) on November 7, 2024.
[0007] In another aspect, the present disclosure provides a method for inducing mutation and breeding of the aforementioned Kluyveromyces marxianus, comprising the following steps:
[0008] (1) Establish a microbial mutant library;
[0009] (2) Preliminary screening of the bacterial cells obtained in step (1);
[0010] (3) The genetic stability of the mutant dominant bacteria was verified to obtain a yeast mutant strain with high RNA content.
[0011] In another aspect, the present disclosure provides a microbial agent containing the aforementioned Kluyveromyces marxianus.
[0012] In another aspect, the present disclosure provides a method for producing RNA, comprising adding the aforementioned Kluyveromyces marxianus to a culture medium and fermenting to produce RNA.
[0013] In another aspect, the present disclosure provides uses of the aforementioned Kluyveromyces marxianus, the aforementioned fermentation product, or the aforementioned microbial agent in the preparation of foods, pharmaceuticals, health products, and agricultural products.
[0014] The beneficial effects of this application are at least as follows:
[0015] The present invention provides a Kluyveromyces marxianus with high nucleic acid content, which is classified and named Kluyveromyces marxianus ( Kluyveromyces marxianus ) CJA0022, with a deposit number of CCTCC No. M 20242475. Its single-cell nucleic acid content can reach 15%, and it has a high growth rate and stable inheritance. The Kluyveromyces marxianus strain disclosed in this application has a broad foundation for the production of yeast nucleic acid products. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 This is the growth curve of the Kluyveromyces marxianus mutant strain CJA0022 in YPD medium.
[0017] Figure 2 is the nucleic acid content of the Kluyveromyces marxianus mutant strain CJA0022. DETAILED DESCRIPTION
[0018] I. Terminology
[0019] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present disclosure belongs.
[0020] The term "and / or" should be understood to mean either one or both of the alternatives.
[0021] The terms "comprising" or "including" generally imply the inclusion of specifically stated features, but not the exclusion of other elements.
[0022] The term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.
[0023] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It should also be understood that, due to deliberate or unintentional mutations, all progeny may not be precisely identical in terms of DNA content. Mutant progeny that possess the same function or biological activity as that screened for in the originally transformed cell are included. Where a different designation is intended, this is clear from the context.
[0024] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and where it does not.
[0025] The term "fermentation" in this application refers to the process in which Kluyveromyces marxianus produces the bacteria itself, or direct metabolites or secondary metabolites through its life activities under aerobic conditions.
[0026] The term "single cell protein" (SCP) refers to a protein obtained from and / or derived from a (single-cell) microorganism. Thus, a single cell protein may refer to a protein purified and / or isolated from, for example, a cell culture of a microorganism. Additionally or alternatively, a single cell protein may refer to a microbial protein that is a dead stem cell of a microorganism. Thus, a "single cell protein product" may or may not contain one or more selected from the group consisting of: intact (single-cell) microbial cells, disrupted (single-cell) microbial cells, an isolated protein obtained from one or more (single-cell) microorganisms, an isolated protein derived from one or more (single-cell) microorganisms, a purified protein obtained from one or more (single-cell) microorganisms, and a purified protein derived from one or more (single-cell) microorganisms. While the (single-cell) microorganism may be a bacterium, a fungus (such as yeast), and / or an algae, according to the present invention, the (single-cell) microorganism is Kluyveromyces marxianus. Single-cell protein products from Kluyveromyces marxianus offer the advantage of providing a relatively high protein content, while at the same time said products can be produced on an industrial scale at relatively low costs, independently of seasonal effects and with relatively low harvesting efforts.
[0027] The present application does not limit the method for collecting the bacteria, and conventional methods in the art, such as centrifugation and filtration, can be used. The present application does not limit the method for separation and purification, and conventional methods in the art, such as precipitation, membrane separation, filtration, flocculation, etc., can be used.
[0028] II. Detailed Description of Examples
[0029] In one aspect, the present disclosure provides a high nucleic acid content Kluyveromyces marxianus, which is classified as Kluyveromyces marxianus ( Kluyveromyces marxianus ) CJA0022, its deposit number is CCTCC No.M20242475.
[0030] In another aspect, the present disclosure provides a method for inducing mutation and breeding of the aforementioned Kluyveromyces marxianus, comprising the following steps:
[0031] (1) Establish a microbial mutant library;
[0032] (2) Preliminary screening of the bacterial cells obtained in step (1);
[0033] (3) The genetic stability of the mutant dominant bacteria was verified to obtain a yeast mutant strain with high RNA content.
[0034] In some embodiments, the establishment of the microbial mutant library adopts chemical mutagenesis, physical mutagenesis, or a combination of chemical mutagenesis and physical mutagenesis.
[0035] In some embodiments, the chemical mutagen is one or a combination of two or more of ethyl methanesulfonate, diethyl sulfate, and sodium azide.
[0036] In some embodiments, the chemical mutagen is present at a concentration of 5% to 20% (w / v). In some embodiments, the chemical mutagen is present at a concentration of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.
[0037] In some embodiments, the chemical mutagen is applied for 20-60 min. In some embodiments, the chemical mutagen is applied for 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min.
[0038] In some embodiments, the physical mutagenesis is one or a combination of two or more of ultraviolet radiation and atmospheric pressure room temperature plasma (ARTP).
[0039] In some embodiments, the physical mutagenesis duration is 30s-100s.
[0040] In some embodiments, the atmospheric pressure room temperature plasma has an operating power of 80-120 W and a gas flow rate of 8-15 L / min.
[0041] In some embodiments, the atmospheric pressure room temperature plasma has an operating power of 100 W and a gas flow rate of 10 L / min.
[0042] In some embodiments, the ultraviolet radiation has an effective power of 15W-30W and an irradiation distance of 20-30cm.
[0043] In some embodiments, the ultraviolet radiation has an effective power of 25 W and an irradiation distance of 25 cm.
[0044] In some embodiments, the initial screening in step (2) is to statically culture the bacteria in a YPD solid culture medium containing a nucleic acid inhibitor.
[0045] In some embodiments, the nucleic acid inhibitor is one or a combination of two or more of 6-azauracil, 8-azaguanine, cycloheximide, 6-mercaptopurine, diaminopurine, 5-bromouracil, and 5-fluorouracil.
[0046] In some embodiments, the inhibitor is at a concentration of 50-1000 mg / L.
[0047] In some embodiments, the pH of the culture medium is 5.0-6.0.
[0048] In some embodiments, the culturing temperature is 28-32°C.
[0049] In some embodiments, the culturing time is 24-72 hours.
[0050] In another aspect, the present disclosure provides a fermentation product of the aforementioned Kluyveromyces marxianus.
[0051] In another aspect, the present disclosure provides a microbial agent containing the aforementioned Kluyveromyces marxianus or the aforementioned fermentation product.
[0052] In another aspect, the present disclosure provides a method for producing RNA, comprising adding the aforementioned Kluyveromyces marxianus to a culture medium and fermenting to produce RNA.
[0053] In some embodiments, the culture medium is YPD medium.
[0054] In some embodiments, the inoculation amount of the strain produced by the fermentation is 0.05%-20%. In some embodiments, the inoculation amount of the strain produced by the fermentation is 2%.
[0055] In some embodiments, the fermentation temperature is 25-45°C.
[0056] In some embodiments, the fermentation culture time is 24-60 h.
[0057] In another aspect, the present disclosure provides uses of the aforementioned Kluyveromyces marxianus, the aforementioned fermentation product, or the aforementioned microbial agent in the preparation of foods, pharmaceuticals, health products, and agricultural products.
[0058] In the following detailed description of the embodiments of the present disclosure, reference is made to the accompanying drawings. Like reference numerals in the drawings indicate like elements, and specific embodiments in which the present disclosure can be practiced are shown in the drawings by way of illustration. These embodiments are described in sufficient detail to enable those skilled in the art to implement the present disclosure. In other cases, well-known processes, structures, and techniques are not shown in detail to avoid confusing the understanding of this specification. Therefore, the following detailed description should not be understood as limiting, and the technical solutions of the present disclosure are limited only by the appended claims.
[0059] Example
[0060] A further understanding of the present disclosure may be obtained by reference to some of the specific examples provided herein. These examples are intended to illustrate the present disclosure only and are not intended to limit the scope of the present disclosure in any way. Obviously, various modifications and variations may be made to the present disclosure without departing from the essence of the present disclosure, and therefore, such modifications and variations are also within the scope of protection claimed in this application.
[0061] Example 1: Mutagenesis and screening of high-nucleic acid Kluyveromyces marxianus strains
[0062] 1. UV mutagenesis
[0063] (1) Using Kluyveromyces marxianus CJ3113 as the starting strain, the starting strain was inoculated into sterile YPD liquid medium and cultured for 2 generations at 30°C; then the stably growing strain was inoculated into sterile liquid YPD medium and cultured for 2 generations to reach a normal growth cycle and set aside.
[0064] (2) Preparation of starting strain cell suspension
[0065] Take 2 mL of the Kluyveromyces marxianus bacterial suspension in the logarithmic growth phase in step (1) and centrifuge at 4000 r / min for 5 minutes; discard the supernatant, resuspend the bacteria in sterile saline, shake gently and wash the bacteria, centrifuge at 4000 r / min for 5 minutes, repeat this step twice; discard the supernatant, add sterile saline and vortex to mix the bacteria to prepare a bacterial suspension. Use the microscopic counting method to adjust the cell concentration to 10 7 CFU / mL.
[0066] (3) Ultraviolet mutagenesis of bacteria
[0067] 3 mL of the prepared bacterial suspension was transferred to a sterile plate with a diameter of 9 cm and spread until the liquid layer reached a thickness of about 0.4 cm. The UV mutagenesis was carried out with a UV lamp power of 25 W, an irradiation time of 100 s, and an irradiation distance of 25 cm. In the dark, 1 mL of the bacterial suspension irradiated by the UV lamp was aspirated and diluted by a ten-fold gradient dilution method. The above 10 4 ~10 6 Spread 0.1 mL of each of the three dilutions onto YPD plates supplemented with a nucleic acid inhibitor and incubate at 30°C for 2-4 days. For initial screening, select 10 colonies with larger phenotypes and inoculate them into YPD liquid medium (1% yeast extract, 2% peptone, and 2% glucose) supplemented with a nucleic acid inhibitor. Repeat the screening in shake flasks. Select dominant mutagenic strains with large colonies and rapid growth in shake flasks, and strains that can be stably propagated for more than three generations in YPD liquid medium supplemented with a nucleic acid inhibitor. Repeat the above steps to screen, eliminating strains with degenerated strains and decreased growth rates, and retaining the primary mutagenic strains with rapid growth and stable genetics.
[0068] 2. Chemical mutagenesis
[0069] The primary mutagenic strain obtained by UV mutagenesis was activated and cultured in YPD liquid medium. Yeast with OD = 1 was taken, centrifuged and the supernatant was discarded. EMS (ethyl methanesulfonate) was added at a final concentration of 5.0%, 7.5%, 10.0%, 12.5%, and 15.0% (w / v), respectively. The mixture was shaken at 30°C and 180 r / min for 30 min. 1.0 mL of the treated solution was quickly added to 1.0 mL of 25% (V / V) sodium thiosulfate solution to terminate the reaction. 1 mL of the treated bacterial solution was taken and diluted by a ten-fold gradient dilution method. The above 10 4 ~10 6Spread 0.1 mL of each of the three dilutions onto YPD solid plates supplemented with a nucleic acid inhibitor and incubate at 30°C for 2-4 days. Perform a preliminary screening by selecting 10 colonies with larger phenotypes and inoculating them into YPD liquid medium supplemented with a nucleic acid inhibitor. Repeat the screening by shake flask fermentation. Select dominant mutagenic strains with large colonies and rapid growth in shake flasks, and strains that can be stably propagated for more than three generations in YPD liquid medium supplemented with a nucleic acid inhibitor. Repeat the above steps for screening, eliminating strains with degenerated strains and decreased growth rates, and retaining the primary mutagenic strains with rapid growth and stable inheritance.
[0070] 3. Atmospheric Pressure Room Temperature Plasma (ARTP) Mutagenesis
[0071] (1) The second-generation mutant strain obtained by chemical mutagenesis was activated and cultured in YPD liquid medium, centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the cells were washed twice with sterile saline and then resuspended in buffer.
[0072] (2) Drop the bacterial solution onto the sterilized slide and use the ARTP mutagenizer to perform ARTP mutagenesis. Set the power to 100 W, the air flow to 10 L / min, and the time to perform mutagenesis for 60 s. After the mutagenesis is completed, place the slide into 1 mL of sterile saline for gradient dilution and select 10 1 ~10 3 Spread onto YPD solid plates supplemented with nucleic acid inhibitors and incubate at 30°C for 2-4 days. Perform a preliminary screening by selecting 10 colonies with larger phenotypes and inoculating them individually into YPD liquid medium supplemented with nucleic acid inhibitors. Repeat the screening with shake flask fermentation. Select dominant mutagenic strains with large colonies and rapid growth in shake flasks, and strains that can be stably propagated for more than three generations in YPD liquid medium supplemented with nucleic acid inhibitors. Repeat the above steps for screening, eliminating strains with degenerated strains and decreased growth rates, and retaining the primary mutagenic strains with rapid growth and stable inheritance.
[0073] (3) Select a single colony obtained in step (2) and inoculate it into YPD liquid culture medium, culture it at a constant temperature of 30°C for 18-28 hours, and determine the bacterial nucleic acid content by spectrophotometry.
[0074] Finally, a mutant strain with the highest nucleic acid content was screened and deposited in the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China. The deposit date was November 7, 2024, the strain deposit number was CCTCC No. M 20242475, and the classification name was Kluyveromyces marxianus CJA0022.
[0075] Example 2: Strain fermentation
[0076] 1) The mutant Kluyveromyces marxianus strain CJA0022 obtained in Example 1 of the present application and the starting Kluyveromyces marxianus strain CJ3113 (as a control strain) were respectively plated and cultured on YPD solid medium (1% yeast extract, 2% peptone, 2% glucose, and 2% agar) at 30°C for 48 hours;
[0077] 2) Pick a single colony obtained in step 1) and activate it in 5 mL of YPD liquid medium at 30°C for 32 hours.
[0078] 3) The bacterial solution obtained in step 2) was expanded in 50 mL of YPD liquid medium and cultured at 30°C for 32 hours;
[0079] 4) Inoculate the bacterial solution obtained in step 3) into fresh YPD medium for shake flask fermentation at a 2% inoculum volume of 200 / 500 mL; incubate in a shaker at 30°C and 200 rpm for 32 hours;
[0080] 5) Collect the cells by centrifugation, wash them twice with water and then freeze-dry them.
[0081] Example 3: Growth curve determination
[0082] Strain activation: Pick a single colony of the mutant strain CJA0022 to be tested on a YPD solid plate, inoculate it into a test tube containing 10 mL of YPD liquid medium and culture it overnight with shaking at 30°C and a shaking speed of 200 rpm. The control strain is the wild-type starting strain CJ3113.
[0083] Strain inoculation: The next day, according to the initial OD 600nm =0.1, inoculate the overnight activated Kluyveromyces marxianus mutant strain and the control strain into a 100 / 250 mL Erlenmeyer flask of growth curve test medium (YPD medium), and culture at 30°C with a shaking speed of 200 rpm.
[0084] Growth curve: Culture for a total of 35 h, and take samples every 4-8 h to measure the OD of the growing strain 600nm value and draw its growth curve.
[0085] Growth curves such as Figure 1 As shown in the figure, compared with the starting strain CJ3113, the growth rate and final biomass of CJA0022 were significantly improved, indicating that the mutant strain of Kluyveromyces marxianus had a significant advantage in biomass under this culture condition.
[0086] Example 4: Nucleic acid content detection
[0087] 1. Reagents
[0088] 0.5N perchloric acid (HClO4): Add 21.5 mL of 70% (or 22.1 mL of 68%) perchloric acid to 400 mL of distilled water, and then dilute to 500 mL with distilled water.
[0089] 0.25N perchloric acid (HClO4): Take 250mL of 0.5N perchloric acid (HClO4) and dilute it to 500mL with distilled water.
[0090] Where N is the equivalent concentration.
[0091] 2. Equipment
[0092] (1) A microgram-grade balance; (2) A 4000 rpm centrifuge capable of separating yeast; (3) Centrifuge tubes with a capacity of at least 10 mL; (4) A spectrophotometer (260 nm); (5) A 70°C hot water bath; (6) A 4°C cold water bath; (7) 10 mL and 1 mL pipettes; and (8) A 100 mL volumetric flask.
[0093] 3. Detection method:
[0094] 1. Prepare freeze-dried yeast powder. Weigh 0.06-0.15g of yeast and add it to a centrifuge tube.
[0095] 2. Add 8 mL of cold 0.25 N HClO4 to the centrifuge tube and incubate in a 4°C water bath for 15 minutes.
[0096] 3. Centrifuge at 4000 rpm for 10 minutes.
[0097] 4. Gently pour off the material floating on the surface, add 45mL of 0.5N HClO and shake to mix. Incubate in a 70°C water bath for 15 minutes, shaking every 3-4 minutes. Centrifuge at 4000 rpm for 10 minutes, aspirate 1mL of the supernatant, and dilute to 100mL with distilled water.
[0098] 5. Measure the absorbance at 260 nm and use distilled water as a blank control.
[0099] 4. The calculation formula is:
[0100] RNA content in dry yeast / %=(A*V) / (M*32)*N*100
[0101] Where:
[0102] A—absorbance value at 260nm;
[0103] V—volume of 0.5N HClO4 solution added, mL;
[0104] N—dilution multiple;
[0105] M—dry weight, mg;
[0106] 32—The unit light absorption value in the literature.
[0107] The results of nucleic acid content test are shown in Figure 2 The nucleic acid content of the composite mutagenesis strain CJA0022 of the present application was increased from 7.9% of the wild-type starting strain to 15%, and the effect was quite significant.
[0108] In summary, the Kluyveromyces marxianus mutant strain CJA0022 provided in the present application has a faster growth rate and a higher nucleic acid yield, and has a high application value in the production of subsequent products.
[0109] The above-described embodiments merely represent several implementation methods of the present disclosure. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person of ordinary skill in the art could make various modifications and improvements without departing from the spirit of the present disclosure, all of which fall within the scope of protection of the present disclosure. Therefore, the scope of protection of the present patent shall be determined by the appended claims.
Claims
1. A high nucleic acid content Kluyveromyces marxianus yeast, which is classified as Kluyveromyces marxianus ( Kluyveromyces marxianus ) CJA0022, its deposit number is CCTCC No. M 20242475.
2. A microbial agent containing the Kluyveromyces marxianus according to claim 1.
3. A method for producing RNA, characterized in that The Kluyveromyces marxianus according to claim 1 is added to a culture medium to ferment and produce RNA.
4. The method according to claim 3, wherein: The culture medium is YPD culture medium.
5. The method according to claim 3, wherein: The inoculation amount of the strain for fermentation production is 0.05%-20%.
6. The method according to claim 5, wherein: The inoculation amount of the strain for the fermentation production is 2%.
7. The method according to claim 3, wherein: The fermentation temperature is 25-45°C.
8. The method according to claim 3, wherein: The fermentation culture time is 24-60h.
9. Use of the Kluyveromyces marxianus according to claim 1 or the microbial agent according to claim 2 in the preparation of food or agricultural products.
10. Use of the Kluyveromyces marxianus according to claim 1 or the microbial agent according to claim 2 in the preparation of health products.