Saccharomyces cerevisiae mutant strain with high yield of S-adenosine-L-methionine as well as construction method and application thereof

Abstract

The invention belongs to the field of microbial breeding and fermentation engineering, and particularly relates to a saccharomyces cerevisiae mutagenesis strain with high yield of S-adenosyl-L-methionine as well as a mutagenesis method and application of the saccharomyces cerevisiae mutagenesis strain. Saccharomyces cerevisiae is induced to generate genetic diversity through multiple rounds of ultraviolet mutagenesis, and high-throughput screening is performed in combination with strain color phenotypic difference; and the mutant strain HY2402-Z18 with high yield of S-adenosine-L-methionine and genetic stability is obtained. The SAM yield of the mutagenic strain is increased by 40.1% compared with that of an original strain, the SAM yield reaches 13.44 g / L when the mutagenic strain is fermented for 60 h in a 5 L fermentation tank system, the unit yield reaches 0.156 g / L / OD, and the methionine conversion rate is 75%. After thalli are collected through centrifugation of fermentation liquor, an S-adenosine-L-methionine product with the purity of 98%-100% can be obtained through purification of ion exchange resin, no by-products are accumulated, the yield, the substrate conversion rate and the production efficiency of the S-adenosine-L-methionine can be remarkably improved when the mutagenesis strain is used for fermentation production, and the production cost is reduced. The production cost is effectively reduced, the product purity and quality are improved, and the method is suitable for industrial large-scale production of SAM.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and relates to a high-yielding S-adenosyl-L-methionine-producing Saccharomyces cerevisiae mutant strain, its mutagenesis method and application, and in particular, to construct a method for breeding high-yielding S-adenosyl-L-methionine-producing Saccharomyces cerevisiae based on ultraviolet mutagenesis and high-throughput screening. Background Technology

[0002] S-Adenosine-L-methionine (SAM) is a naturally occurring bioactive substance widely found in animals, plants, and microorganisms. SAM is a substrate for many enzyme-catalyzed reactions. It not only provides methyl groups for many methylation reactions but also acts as a precursor in the biosynthesis of polyamines, spermine and spermine, nicotinamide, and the gaseous plant hormone ethylene. It is closely linked to the biosynthesis of proteins, nucleic acids, neurotransmitters, phospholipids, and vitamins.

[0003] SAM production can be achieved through chemical synthesis, enzymatic catalysis, and microbial fermentation. Chemical synthesis is not widely used due to difficulties in product purification, low yield, and environmental pollution. Compared with chemical synthesis, enzymatic catalysis produces products that are easier to purify, have higher purity, and exhibit greater stability of methionine. However, the low content and difficulty in purifying and preserving SAM synthase in organisms, coupled with the high cost of the substrate ATP, limit enzymatic catalysis to laboratory-scale production and hinder industrialization. Microbial fermentation is currently the most commonly used SAM production method due to its low production cost and suitability for industrial production. It is also the main production method for SAM. However, existing technologies still face yield bottlenecks. For example, the genetically engineered strain reported in patent CN115820443A, although achieving a shake flask yield of 1.73 g / L and a 5L fermenter yield of 7.43 g / L through SOD1 gene knockout, still falls short of industrial-scale requirements. Besides genetic engineering, existing technologies also attempt to irrationally modify Saccharomyces cerevisiae through UV mutagenesis and chemical mutagenesis to increase SAM production. However, existing mutagenesis technologies have significant drawbacks: they do not optimize parameters for the biosynthetic characteristics of S-adenosyl-L-methionine, and they do not consider the thick cell wall and strong resistance to mutagens in yeast, leading to insufficient mutagenesis intensity or excessive cell damage; they lack specific screening methods for S-adenosyl-L-methionine, relying mainly on strain morphology screening, which easily misses high-yielding mutants, and they have not systematically verified the S-adenosyl-L-methionine synthesis stability of mutants, making them difficult to apply to industrial production.

[0004] Therefore, there is an urgent need to develop an efficient synergistic mutagenesis method targeting the synthetic characteristics of yeast S-adenosine-L-methionine to achieve targeted screening of high-yield and stable mutant strains. Summary of the Invention

[0005] The purpose of this invention is to provide a high-yielding S-adenosyl-L-methionine-producing Saccharomyces cerevisiae mutant strain, its mutagenesis method, and its application. The high-yielding S-adenosyl-L-methionine (SAM) Saccharomyces cerevisiae mutant strain is obtained through mutagenesis and high-throughput screening.

[0006] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a high-yielding S-adenosyl-L-methionine-producing Saccharomyces cerevisiae mutant strain, which is deposited at the China Center for Type Culture Collection on September 19, 2025, with accession number CCTCC NO:M 20252069.

[0008] Secondly, the present invention provides a method for constructing a *Saccharomyces cerevisiae* mutant strain, the method comprising:

[0009] (1) Using Saccharomyces cerevisiae HY2402-Z1 as the starting strain, a bacterial suspension was prepared and the bacterial suspension was subjected to ultraviolet mutagenesis to obtain a mutagenized bacterial suspension;

[0010] (2) Spread the mutagenized bacterial suspension obtained in step (1) onto the screening medium and incubate at 30°C for 3-4 days. Screen out the initial positive strains based on the differences in colony color phenotype.

[0011] (3) The positive strains obtained in step (2) were inoculated into YPD liquid culture medium and cultured under shaking conditions at 30℃ and 200-300rpm. By measuring the yield of S-adenosine-L-methionine, a high-yielding Saccharomyces cerevisiae mutant strain was obtained.

[0012] S-Adenosyl-L-methionine (SAM) is the main methyl donor for ergosterol synthesis in yeast and is consumed in large quantities. Nystatin can bind to ergosterol on the yeast cell membrane, leading to the loss of the ergosterol production pathway and altered cell membrane permeability. Therefore, nystatin can be used to breed high-yielding S-Adenosyl-L-methionine-producing mutant strains of *Saccharomyces cerevisiae*. The sulfur assimilation pathway may be closely related to the biosynthesis of S-Adenosyl-L-methionine in *Saccharomyces cerevisiae*. High concentrations of S-Adenosyl-L-methionine regulate Met4p activity and inhibit the expression of genes related to the sulfur assimilation pathway. Reduced sulfur assimilation leads to a corresponding decrease in H2S production, indicating that the biosynthesis of S-Adenosyl-L-methionine can negatively regulate H2S formation. Pb... 2+ Can be used with S 2-The presence of a distinct black precipitate of PbS allows for visual differentiation. Based on this phenomenon, this invention establishes a high-throughput, rapid screening method for *Saccharomyces cerevisiae* mutant strains by adding lead acetate to the screening medium.

[0013] Preferably, in step (1), the total number of colonies in the starting strain suspension is 10. 6 -10 8 CFU / mL.

[0014] Preferably, in step (1), the ultraviolet mutagenesis is performed by irradiation with a 15W ultraviolet lamp, the irradiation distance is 30cm, and the irradiation time is 30-240 s.

[0015] Preferably, the screening medium comprises the following components: glucose 15-25 g / L, ammonium sulfate 5-15 g / L, yeast extract 1-5 g / L, methionine 0.1-0.5 g / L, lead acetate 1-5 g / L, agar powder 10-20 g / L, and nystatin 1-20 μg / mL.

[0016] More preferably, the screening medium comprises the following components: 20 g / L glucose, 10 g / L ammonium sulfate, 3 g / L yeast extract, 0.2 g / L methionine, 1 g / L lead acetate, 15 g / L agar powder, and 1-20 μg / mL nystatin.

[0017] The *Saccharomyces cerevisiae* mutant strain screened using the construction method of the present invention can produce at least 2 g / L of S-adenosyl-L-methionine in 48 hours when shake-flask fermentation is carried out in a fermentation medium containing L-methionine.

[0018] Thirdly, the present invention provides an application of the described Saccharomyces cerevisiae mutant strain in the fermentation production of S-adenosine-L-methionine.

[0019] Fourthly, the present invention provides a fermentation production method for S-adenosyl-L-methionine, the production method comprising: inoculating the mutant strain of Saccharomyces cerevisiae into a fermentation medium for fermentation culture, and then separating and purifying it by ion exchange resin to obtain the target product S-adenosyl-L-methionine.

[0020] Preferably, the production method includes the following steps:

[0021] Step 1: Inoculate the described Saccharomyces cerevisiae mutant strain into YPD liquid medium and culture it with shaking at 30℃ and 200-250 rpm for 10-20 h to obtain seed culture;

[0022] Step 2: Inoculate the seed culture obtained in Step 1 into the fermentation medium at an inoculation rate of 1-5%, and ferment at 30℃ and 200-250 rpm for 36-72 h, and collect the fermentation broth.

[0023] Step 3: Centrifuge the fermentation broth obtained in Step 2 and collect the cell cells;

[0024] Step 4: Break the bacterial cells obtained in Step 3, centrifuge to collect the supernatant, and use ion exchange resin to separate and purify the supernatant. Collect the eluent rich in S-adenosine-L-methionine, concentrate and dry it to obtain S-adenosine-L-methionine.

[0025] Preferably, the fermentation medium consists of the following components: glucose 30 g / L, yeast extract 3 g / L, ammonium sulfate 5 g / L, dipotassium hydrogen phosphate 5 g / L, potassium dihydrogen phosphate 10 g / L, manganese sulfate heptahydrate 0.1 g / L, zinc sulfate heptahydrate 0.1 g / L, magnesium sulfate 0.2 g / L, calcium chloride 0.1 g / L, copper sulfate 0.0016 g / L, and sodium citrate 0.1 g / L.

[0026] Preferably, in step 2, the fermentation culture is divided into shake flask culture or fermenter culture.

[0027] Preferably, when using shake flask culture, 4 mL of methionine at a concentration of 18 g / L is added after 24 h of fermentation.

[0028] Preferably, when culturing in a fermenter, the pH of the fermentation system is stabilized at 5.8 and the dissolved oxygen is maintained at 20-50% by automatically adding ammonia water.

[0029] More preferably, when cultured in a fermenter, the fermentation temperature is controlled at 30°C, the pH is stabilized at 5.8 by automatic ammonia addition, dissolved oxygen is maintained at 30%, and OD... 600 Once the protein level reaches 60, methionine supplementation begins, every 4 hours. For the first 5 supplements, add 5.2g of methionine, and then add 2.6g of methionine each time thereafter.

[0030] Preferably, step 4 includes:

[0031] Bacterial cells were broken by repeated freeze-thaw cycles to release intracellular components and obtain a bacterial mixture.

[0032] Centrifuge the bacterial cell mixture and collect the supernatant (i.e., the separation solution containing SAM).

[0033] The separation solution was loaded with ion exchange resin at a rate of 1 mL / min to allow SAM to undergo ion exchange adsorption with the resin, resulting in resin adsorbed with S-adenosylmethionine.

[0034] The solution was eluted with 0.2-0.5 mol / L sulfuric acid at a flow rate of 1 mL / min. The eluent containing SAM was collected, concentrated and dried to obtain S-adenosine-L-methionine.

[0035] Preferably, the ion exchange resin is selected from any one of the following: weak acid cation exchange resin JK110, strong acid cation exchange resin ZGC107, macroporous weak acid cation exchange resin ZGC258, and HZG1047M.

[0036] This invention provides an application of the described *Saccharomyces cerevisiae* mutant strain in the preparation of S-adenosyl-L-methionine drugs or nutritional supplements. Specifically, the *Saccharomyces cerevisiae* mutant strain obtained by this invention can efficiently express S-adenosyl-L-methionine and can be applied to three core scenarios: liver protection, mood regulation, and joint health, while also covering some metabolic auxiliary improvement needs.

[0037] Compared with existing technologies, the beneficial effects of this invention are mainly reflected in the following aspects: Through multiple rounds of UV mutagenesis combined with a specific screening strategy, a high-yield SAM strain, HY2402-Z18, was successfully obtained, with its SAM yield increasing by 40.1% compared to the starting strain. In a 5L fermenter system, after 61.4 hours of fermentation, the SAM yield of this strain reached 13.44 g / L, with significantly better unit yield (0.156 g / L / OD) and methionine conversion rate (75%) than existing technologies. After centrifugation to collect the cells, the fermentation broth was purified using ion exchange resin, resulting in SAM purity of 98-100%, with no accumulation of byproducts such as adenosine, S-adenosylmethionine, decarboxylated S-adenosylmethionine, and S-adenosine-L-homocysteine. The purification efficiency is significantly improved compared to traditional processes. The application of this mutant strain can greatly improve SAM production efficiency while reducing raw material consumption and purification costs, resulting in low industrial production costs. Furthermore, the standardized process for efficient mutagenesis and selection of high-yield SAM strains provided by this invention, including optimization of UV mutagenesis parameters, design of specific screening plates, and control of fermentation process, can provide technical reference for the selection of other high-yield amino acid strains and has broad industrial application prospects. Attached Figure Description

[0038] Figure 1 To screen for different colony colors in the plate.

[0039] Figure 2 The results show the yield of S-adenosine-L-methionine in shake-flask re-screening of UV-mutated strains.

[0040] Figure 3 The growth curve of the mutant strain HY2402-Z18 is shown.

[0041] Figure 4 The growth curve of the passaged strain HY2402-Z18-30 is shown.

[0042] Figure 5 The genetic stability of the mutant strain HY2402-Z18.

[0043] Figure 6 Results of S-adenosine-L-methionine production in the fermenter of mutant strain HY2402-Z18.

[0044] Figure 7 High-performance liquid chromatography (HPLC) image of S-adenosine-L-methionine prepared from the mutant strain HY2402-Z18. Detailed Implementation

[0045] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0046] The brewing yeast HY2402-Z1 (also named brewing yeast ZJS10041) used in this invention is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 2020495, deposit date July 21, 2021, address: Wuhan University, Wuhan, China, postcode 430072. It has been disclosed in patent application number 202111307689.9 and was purchased from the China Center for Type Culture Collection.

[0047] YPD liquid medium: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L.

[0048] YPD solid medium: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L, yeast extract 15 g / L.

[0049] Screening medium: glucose 20 g / L, ammonium sulfate 10 g / L, yeast extract 3 g / L, methionine 0.2 g / L, lead acetate 1 g / L, agar powder 15 g / L, and nystatin 1-20 μg / mL.

[0050] Fermentation medium: glucose 30 g / L, yeast extract 3 g / L, ammonium sulfate 5 g / L, dipotassium hydrogen phosphate 5 g / L, potassium dihydrogen phosphate 10 g / L, manganese sulfate heptahydrate 0.1 g / L, zinc sulfate heptahydrate 0.1 g / L, magnesium sulfate 0.2 g / L, calcium chloride 0.1 g / L, copper sulfate 0.0016 g / L, sodium citrate 0.1 g / L.

[0051] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0052] Example 1: Screening of Saccharomyces cerevisiae mutant strains producing high levels of S-adenosine-L-methionine

[0053] (1) Preparation of bacterial suspension: Cryopropyl yeast HY2402-Z1 was streaked onto YPD solid medium and cultured at 30℃ for 3-4 days. Fresh single colonies were inoculated into 10 mL of YPD liquid medium and cultured at 30℃ and 220 rpm for 14 h. Then, 1% of the inoculum was transferred to 50 mL of YPD liquid medium and cultured at 30℃ and 220 rpm until the logarithmic growth phase. After washing three times with sterile physiological saline, the bacterial suspension was resuspended with physiological saline to obtain a total bacterial count of 10. -6 -10 -8 A bacterial suspension of CFU / mL was prepared for use in mutagenesis.

[0054] (2) Parameter settings: The working power of the ultraviolet lamp in the ultraviolet mutation chamber is 15W, and the ultraviolet treatment time gradient is 0, 30, 60, 90, 120, 150, 180, 210 s, 240 s.

[0055] (3) Mutagenesis treatment procedure: Take 5 mL of yeast suspension and transfer it to a sterile plate on a magnetic stirrer. Place the magnetic stirrer 30 cm away from the UV lamp. Perform the treatment according to the above parameters to obtain the mutagenized bacterial suspension; among them, the mutagenesis treatment time of 0 s is the control group, and the mutagenesis treatment times of 30, 60, 90, 120, 150, 180, 210 s, and 240 s are the treatment groups. After mutagenesis, the samples need to be stored in an ice bath in the dark for 2 hours.

[0056] (4) Sample plating: After the control group and treatment group samples were protected from light for 2 hours, 200 μL of each sample was plating onto the screening medium and incubated in a 30℃ incubator for 3 days. The colony count was recorded and the lethality was calculated (lethality = (number of single clones in the control group - number of single clones in the treatment group) / number of single clones in the control group × 100%).

[0057] (5) Plate screening:

[0058] Remove the screening plates with good colony growth. Since a lethality rate of 80-90% makes it easier to screen for effective mutations, plates with a lethality rate of 80-90% were selected for comparison of colony color and size. Compare the color and size of the colonies in the culture medium, and select single bacteria with darker color and larger colonies as the initial positive strains for shake-flask rescreening (refer to...). Figure 1 ).

[0059] (6) Shaking flask and sieving

[0060] The positive strains obtained from the initial screening were inoculated into YPD liquid medium and cultured for 14-16 h. The initial inoculation OD was controlled at 3 and the inoculation was carried out into a 50 mL shake flask fermentation medium. The culture was carried out at 30℃ and 220 rpm for 48 h for shake flask re-screening. 4 mL of 18 g / L methionine solution was added after 24 h of fermentation.

[0061] The results of shake-flask fermentation show (reference) Figure 2 A total of 110 single colonies were selected on screening solid plates. Among them, 21 strains had a SAM yield greater than 2 g / L, which was about 40% higher than the basic strain HY2402-Z1. Among them, strain No. 89 had the highest yield, reaching 2.34 g / L. This mutant strain was named HY2402-Z18.

[0062] Example 2: Stability test of the mutagenized strain

[0063] To verify the genetic stability of the mutant strain HY2402-Z18 obtained through screening, the mutant strain was passaged 30 times on YPD solid medium to obtain the passaged strain HY2402-Z18-30.

[0064] The growth characteristics of the strains before and after subculturing were analyzed by growth curve determination: HY2402-Z18 and HY2402-Z18-30 were cultured simultaneously, and samples were taken every 2 hours to detect their OD. 600 The value was used to characterize the amount of bacterial growth, and the results are shown in [Figure number missing]. Figure 3 , Figure 4 The results showed that the growth dynamics of the two strains were highly consistent. Both entered the rapid growth phase after 12 hours of culture and maintained logarithmic growth from 12 to 18 hours. Furthermore, there was no statistically significant difference in the overall specific growth rate of the strains, indicating that continuous subculturing did not significantly affect the cell growth capacity.

[0065] Based on the growth characteristic analysis results, further shake-flask fermentation experiments were conducted to verify the effect of subculturing on the strain's product synthesis ability. The results are shown in […]. Figure 5 Fermentation data showed that after 48 hours of fermentation, the OD of the mutant strain HY2402-Z18 was... 600The OD value reached 11.53±0.505, the S-adenosine-L-methionine yield was 2.27±0.121 g / L, and the content of each byproduct was less than 0.05 g / L; after 30 subcultures, the passaged strain HY2402-Z17-30, under the same fermentation conditions, showed an OD value of 11.53±0.505 after 48 hours of shake-flask fermentation. 600 The value was 11.93±0.702, the SAM yield was 2.28±0.172 g / L, and the byproduct content was also maintained below 0.05 g / L.

[0066] Based on the growth curves and shake-flask fermentation results, the yield of the target product SAM differed by ≤10% before and after subculturing, and there were no significant fluctuations in cell growth status and byproduct synthesis levels. Therefore, the mutant strain HY2402-Z18 obtained through mutagenesis maintains stable cell growth and S-adenosyl-L-methionine synthesis capabilities during continuous subculturing, demonstrating excellent genetic stability. This meets the requirements for strain stability in the industrial production of S-adenosyl-L-methionine and can be considered a candidate strain for the industrial production of this product.

[0067] Example 3: Fermenter Test of Mutagenic Bacteria

[0068] To further investigate the fermentation performance of the mutant strain HY2402-Z18, fermentation tests were conducted in a 5 L fermenter.

[0069] (1) Preparation of culture medium:

[0070] YPD liquid medium: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L.

[0071] YPD solid medium: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L, yeast extract 15 g / L.

[0072] Fermentation medium: glucose 30 g / L, yeast extract 3 g / L, ammonium sulfate 5 g / L, dipotassium hydrogen phosphate 5 g / L, potassium dihydrogen phosphate 10 g / L, manganese sulfate heptahydrate 0.1 g / L, zinc sulfate heptahydrate 0.1 g / L, magnesium sulfate 0.2 g / L, calcium chloride 0.1 g / L, copper sulfate 0.0016 g / L, sodium citrate 0.1 g / L.

[0073] (2) Fermentation culture in a fermenter:

[0074] ①Strike the mutant strain HY2402-Z18 onto YPD solid medium and incubate at 30℃ for 4 days.

[0075] ②Inoculate well-grown colonies into 50 mL of YPD liquid medium and incubate at 30℃ and 220 rpm for 14 h.

[0076] ③ Transfer the seed culture to a 5 L fermenter containing 2 L of fermentation medium, control the temperature at 30℃, automatically add ammonia water to stabilize the pH at 5.8, maintain dissolved oxygen at around 30%, and continue fermentation for 68 h.

[0077] (3) Isolation and detection of S-adenosine-L-methionine

[0078] (1) Sample preparation: Take 1.0 mL of fermentation broth, centrifuge at 12000 rpm for 5 minutes, discard the supernatant and wash twice with sterile water; then add 200 μL of pure water and 200 μL of ethyl acetate, and shake in a metal bath at 37℃ and 800 rpm for 0.5 h; then add 500 μL of 0.35 mol / L sulfuric acid, and continue shaking at 37℃ and 800 rpm for 1.5 h; then centrifuge at 12000 rpm for 5 minutes, and filter the supernatant through a 0.22 μm filter membrane.

[0079] (2) High performance liquid chromatography detection conditions: C18 column, detection wavelength 260 nm, flow rate 1 ml / min, column temperature 30℃, detection time 20 min; mobile phase is 0.1 mol / L ammonium formate + 0.1% (g / g) sodium octanesulfonate, formic acid to adjust pH 4 as mobile phase A, methanol as mobile phase B, isocratic elution according to A:B=75%:25%, flow rate 1.0 mL / min, column temperature 35℃, detection wavelength 260 nm, injection volume 10 μL.

[0080] Fermentation results show (reference) Figure 6 The mutant strain HY2402-Z18 was fermented in a fermenter for 61.4 h, and the highest yield of S-adenosyl-L-methionine reached 13.44 g / L, with a maximum biomass OD. 600 The yield reached 86.4 g / L / OD, with a unit OD yield of 0.156 g / L / OD and a methionine molar conversion rate of 75%. Using this mutant strain for fermentation to produce S-adenosyl-L-methionine can significantly increase the yield of S-adenosyl-L-methionine and the methionine conversion rate, thereby improving production efficiency and effectively reducing production costs.

[0081] Example 4: SAM separation and purification

[0082] (1) Pretreatment of fermentation broth:

[0083] Take the fermentation broth from Example 3, freeze it overnight at -20°C, then thaw it and cycle it three times to break the yeast cells to obtain a cell mixture.

[0084] The bacterial cell mixture was centrifuged at 10,000 rpm and 10 °C for 10 min, and the supernatant containing SAM was collected. The SAM separation solution was a pale yellow transparent liquid. The initial concentration and yield of SAM were obtained by liquid chromatography detection of the SAM yield in the supernatant and precipitate.

[0085] (2) Ion exchange adsorption and elution:

[0086] ① Resin pretreatment: After repeatedly washing the resin with hot water, pretreatment is carried out according to the following procedure.

[0087] S1: Slowly flow 1N hydrochloric acid through the resin, using approximately 2-3 times the volume of strong acid resin and 3-5 times the volume of weak acid resin, with 1.5 times the resin volume flowing through per hour.

[0088] S2: Rinse with water; the pH of the effluent is around 4.

[0089] S3: Use 1NNaOH to flow through the resin, with the same amount and flow rate as S1.

[0090] S4: Rinse with water until the pH of the effluent is around 10.

[0091] S5: Use 1N hydrochloric acid to convert the resin to H*, the amount of which is 3-5 times the volume of the resin, and the flow rate is the same as in S1.

[0092] S6: After the acid flow is complete, rinse with deionized water until the pH of the effluent is above 6, then it can be put into use.

[0093] ② Adsorption: A supernatant containing SAM, after cell wall disruption and centrifugation, was added to the pretreated resin. The resin was loaded with SAM at a rate of 1 mL / min for adsorption, resulting in resin adsorbed with S-adenosylmethionine. The SAM concentration in the eluent was detected using high-performance liquid chromatography (HPLC). The presence of SAM in the eluent indicated resin saturation. Adsorption was performed using different types of resins, including: weakly acidic cation exchange resin JK110, strongly acidic cation exchange resin ZGC107, macroporous weakly acidic cation exchange resin ZGC258, and HZG1047M.

[0094] (3) Desorption and elution: Elution was performed using 0.2-0.5 mol / L sulfuric acid solution at a flow rate of 1 mL / min. The concentration of SAM in the eluent was detected by high performance liquid chromatography. After adsorption and desorption experiments with the above-mentioned different resins, the purity of the purified SAM was 98-100% based on the area normalization method.

[0095] (4) Concentration and drying:

[0096] ① Sample pretreatment: The eluent needs to be filtered first (using a 0.22-0.45μm filter membrane) to remove particulate matter and avoid contaminating the freeze dryer or clogging the pipes. The sample volume should be controlled at 1 / 3-1 / 2 of the freeze dryer bottle to avoid expansion and overflow after pre-freezing.

[0097] ② Pre-freezing stage: Place the freeze-drying bottle in a -20 degree Celsius freezer and statically freeze for 4-8 hours to ensure that the sample is completely frozen (without liquid flow).

[0098] ③ Drying: The vacuum degree is initially set at 10-30 Pa, and can be reduced to 5-10 Pa after the sample has completely sublimated. The shelf temperature is initially the same as the pre-freezing temperature, and after 1-2 hours, it is gradually increased to 0-10℃ (5-10℃ per hour to avoid melting the sample). The drying time is 8-24 hours. After drying, pure SAM is obtained.

[0099] In summary, this invention induces genetic diversity in *Saccharomyces cerevisiae* through multiple rounds of UV mutagenesis and uses high-throughput screening of the mutant strains based on color differences on screening plates, thereby obtaining a superior strain HY2402-Z18 that is high-yielding and genetically stable in S-adenosyl-L-methionine production. Experimental results show that the SAM yield of this strain is 40.1% higher than that of the original strain. In a 5L fermenter system, after 60 h of fermentation, the SAM yield reaches 13.44 g / L, with a yield of 0.156 g / L / OD and a methionine conversion rate of 75%. After centrifugation to collect the cells, the fermentation broth is purified using ion exchange resin to obtain S-adenosyl-L-methionine product with a purity of 98-100%, without the accumulation of byproducts such as adenosine, S-adenosylmethionine (SAMe), decarboxylated S-adenosylmethionine, and S-adenosyl-L-homocysteine. Fermentation using this high-yield strain can significantly improve the yield, substrate conversion rate, and production efficiency of S-adenosyl-L-methionine, effectively reduce production costs, and improve product purity and quality. It is suitable for the industrial-scale production of S-adenosyl-L-methionine.

[0100] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A high-yielding Saccharomyces cerevisiae mutant strain producing S-adenosyl-L-methionine, characterized in that, The saccharomyces cerevisiae mutant strain, with the accession name Saccharomyces cerevisiae HY2402-Z18, was deposited at the China Center for Type Culture Collection on September 19, 2025, with accession number CCTCC NO:M 20252069.

2. A method for constructing a *Saccharomyces cerevisiae* mutant strain according to claim 1, characterized in that, The construction method includes: (1) Using Saccharomyces cerevisiae HY2402-Z1 as the starting strain, a bacterial suspension was prepared and the bacterial suspension was subjected to ultraviolet mutagenesis to obtain a mutagenized bacterial suspension; (2) Spread the mutagenized bacterial suspension obtained in step (1) onto the screening medium and incubate at 30°C for 3-4 days. Screen out the initial positive strains based on the differences in colony color phenotype. (3) The positive strains obtained in step (2) were inoculated into YPD liquid medium and cultured with shaking at 30℃ and 200-300 rpm. By measuring the yield of S-adenosine-L-methionine, a high-yielding Saccharomyces cerevisiae mutant strain was obtained.

3. The construction method according to claim 2, characterized in that, In step (1), the total number of colonies in the starting strain suspension is 10. 6 -10 8 CFU / mL.

4. The construction method according to claim 2, characterized in that, In step (1), the ultraviolet mutagenesis uses 15W ultraviolet light, the irradiation distance is 30cm, and the irradiation time is 30-240s.

5. The construction method according to claim 2, characterized in that, The screening medium contains the following components: glucose 15-25 g / L, ammonium sulfate 5-15 g / L, yeast extract 1-5 g / L, methionine 0.1-0.5 g / L, lead acetate 1-5 g / L, agar powder 10-20 g / L, and nystatin 1-20 μg / mL.

6. The use of the *Saccharomyces cerevisiae* mutant strain according to claim 1 or the *Saccharomyces cerevisiae* mutant strain obtained by the construction method according to any one of claims 2-5 in the fermentation production of S-adenosine-L-methionine.

7. A fermentation method for producing S-adenosine-L-methionine, characterized in that, The production method includes the following steps: after the mutant strain of Saccharomyces cerevisiae is cultured by seed, it is inoculated into a fermentation medium for fermentation culture, and then separated and purified by ion exchange resin to obtain the target product S-adenosine-L-methionine.

8. The fermentation production method according to claim 7, characterized in that, Includes the following steps: Step 1: Inoculate the *Saccharomyces cerevisiae* mutant strain described in claim 1 or the *Saccharomyces cerevisiae* mutant strain obtained by the construction method described in any one of claims 2-5 into YPD liquid medium and culture it with shaking at 30°C and a rotation speed of 200-250 rpm for 10-20 h to obtain seed culture. Step 2: Inoculate the seed culture obtained in Step 1 into the fermentation medium at an inoculation rate of 1-5%, and ferment at 30℃ and 200-250 rpm for 36-72 h, and collect the fermentation broth. Step 3: Centrifuge the fermentation broth obtained in Step 2 and collect the bacterial cells; Step 4: Break the bacterial cells obtained in Step 3, centrifuge to collect the supernatant, and use ion exchange resin to separate and purify the supernatant. Collect the eluent rich in S-adenosine-L-methionine, concentrate and dry it to obtain S-adenosine-L-methionine.

9. The fermentation production method according to claim 8, characterized in that, The fermentation medium consists of the following components: glucose 30 g / L, yeast extract 3 g / L, ammonium sulfate 5 g / L, dipotassium hydrogen phosphate 5 g / L, potassium dihydrogen phosphate 10 g / L, manganese sulfate heptahydrate 0.1 g / L, zinc sulfate heptahydrate 0.1 g / L, magnesium sulfate 0.2 g / L, calcium chloride 0.1 g / L, copper sulfate 0.0016 g / L, and sodium citrate 0.1 g / L.

10. The fermentation production method according to claim 8, characterized in that, In step 2, the fermentation culture is divided into shake flask culture or fermenter culture: when it is shake flask culture, 4 mL of methionine with a concentration of 18 g / L is added after 24 h of fermentation; when it is fermenter culture, the pH of the fermentation system is stabilized at 5.8 and the dissolved oxygen is maintained at 20-50% by automatically adding ammonia water.

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