Engineering bacterium for producing S-ademetionine as well as construction method and application of engineering bacterium

Abstract

The invention provides an engineering bacterium for producing S-adenosylmethionine and a construction method and application thereof, and belongs to the technical field of biology, a saccharomyces cerevisiae engineering bacterium for producing high-yield S-adenosylmethionine is constructed after a glycogen branched chain enzyme GLC3 coding gene in saccharomyces cerevisiae FRD-SK-25 is knocked out, the yield of the S-adenosylmethionine in the engineering bacterium can reach 12.2 g / L, and the engineering bacterium can be used for producing S-adenosylmethionine. And compared with the original strain saccharomyces cerevisiae FRD-SK-25, the yield of the saccharomyces cerevisiae FRD-SK-25 is increased by about 87.7%, and an excellent strain is provided for industrial production of S-ademetionine. In addition, the S-ademetionine-containing supernate obtained through fermentation of the saccharomyces cerevisiae engineering bacteria further has the effects of resisting aging and repairing skin barriers, and a theoretical basis is provided for application of S-ademetionine in the fields of cosmetics, health food and the like.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an engineered bacterium that produces S-adenosylmethionine, its construction method, and its applications. Background Technology

[0002] S-Adenosylmethionine (SAM), also known as S-adenosyl-L-methionine, was first discovered in rat liver by Italian scientists. In vivo, it is synthesized from methionine (Met) and ATP through the catalytic reaction of SAM synthase. SAM participates in the synthesis and metabolism of nucleic acids, proteins, and phospholipids, and is an essential substance for maintaining normal cell function. Clinically, SAM has high medicinal value, primarily used to treat intrahepatic cholestasis. It can also be used to treat viral hepatitis, alcoholic liver disease, improve liver function, alleviate depressive symptoms in patients with depression, and treat rheumatoid arthritis.

[0003] Saccharomyces cerevisiae possesses many excellent characteristics, including a clear genetic background, food-grade safety, strong stress resistance, and easy control of fermentation conditions. However, the intracellular SAM accumulation in traditional Saccharomyces cerevisiae is less than 2% DCW (dry weight of cells), and SAM is highly sensitive to ethanol and osmotic pressure in the later stages of fermentation, making it difficult to achieve high yields. To meet the growing market demand, there is an urgent need to construct a high-yield engineered SAM strain for large-scale industrial production of SAM.

[0004] The invention patent application with publication number "CN105483190A" discloses a method for genetically engineering Saccharomyces cerevisiae to increase the yield of S-adenosine-L-methionine. First, a one-step gene substitution method is used to replace one glycogen branched chain enzyme GLC3 allele on the chromosome of the Saccharomyces cerevisiae strain with the G418 resistance gene. Then, haploids containing only the replaced GLC3 gene are obtained by spore isolation, thus obtaining homozygotes with the GLC3 gene mutation. Fermentation verification in 10L and 500L fermenters showed that the mutant strain produced S-adenosine methionine yields of 7.93 g / L and 8.35 g / L, respectively, which were 15.1% and 24.7% higher than the original strain. The invention patent application with publication number "CN105483153A" discloses a method for improving the production level of S-adenosyl-L-methionine by metabolic engineering modification of Saccharomyces cerevisiae. Utilizing yeast gene knockout technology, the method involves first knocking out the glycogen branched-chain enzyme GLC3 gene and then knocking out the S-adenosyl-methionine decarboxylase SPE2 gene, or first knocking out the SPE2 gene and then knocking out the GLC3 gene. This significantly increases the yield of S-adenosyl-methionine produced by the Saccharomyces cerevisiae strain by up to 55.1%. Combining the above two patent documents, it is known that knocking out the glycogen branched-chain enzyme GLC3 gene alone in Saccharomyces cerevisiae has a limited effect on increasing S-adenosyl-methionine production, only by 15.1% or 24.7%; even when both GLC3 and SPE2 are knocked out simultaneously, the effect on increasing S-adenosyl-methionine production in the Saccharomyces cerevisiae strain is only about 55.1%, with a yield of 10.71 g / L.

[0005] As the outermost and continuously exposed organ of the body, the skin serves as a "firewall" against multiple physical, chemical, and microbial attacks, and is also a core interface for maintaining water-electrolyte balance, immune surveillance, and nerve sensation. The skin aging process can be divided into two main lines based on triggering factors: one is endogenous aging, which gradually manifests over time and accounts for only about 20% of the overall aging load; the other is photoaging induced by ultraviolet radiation (290–400 nm) from sunlight, contributing as much as 80% and exhibiting a "cumulative-amplification" characteristic. UV irradiation instantaneously stimulates the NADPH oxidase and mitochondrial electron leakage pathways, explosively producing reactive oxygen species (ROS), which in turn activates the AP-1 / NF-κB signaling axis, simultaneously upregulating matrix metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2). This cascade not only triggers chronic low-grade inflammation but also leads to cell cycle arrest and increased sensitivity to apoptosis, ultimately resulting in a net loss of type I / III collagen, elastin, and glycosaminoglycans in the extracellular matrix (ECM), leading to wrinkles, sagging skin, and photodamaged age spots. Studies on aging mechanisms have found that autophagy can delay organ function decline and help cells restore metabolic homeostasis by clearing damaged proteins and lipids from UV radiation, thus providing protection for photoaged skin. In summary, if different aging pathways in the skin can be inhibited while simultaneously improving skin cell condition, the aging process can be resisted to a greater extent. Therefore, the search for multi-level and multi-dimensional anti-aging active substances is a current research hotspot. Currently, there is no literature to support the practical application of S-adenosylmethionine in anti-aging and other skincare fields. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an engineered bacterium that produces S-adenosylmethionine, its construction method, and its applications.

[0007] The technical solution of this invention is as follows: An engineered strain of brewer's yeast is a strain of brewer's yeast ( Saccharomyces cerevisiae The glycogen branched chain enzyme GLC3 encoding gene was knocked out in FRD-SK-25. The Saccharomyces cerevisiae FRD-SK-25 was deposited on September 22, 2025 at the China General Microbiological Culture Collection Center, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, accession number: CGMCC No. 35987.

[0008] The nucleotide sequence of the glycogen branched chain enzyme GLC3 encoding gene is shown in SEQ ID NO.1, and the amino acid sequence of the glycogen branched chain enzyme GLC3 is shown in SEQ ID NO.2.

[0009] The method for constructing the engineered Saccharomyces cerevisiae includes the following steps: using the genome of Saccharomyces cerevisiae strain FRD-SK-25 as a template, the left and right homologous arm fragments of glycogen branched chain enzyme GLC3 are amplified by PCR reaction, and the left and right homologous arm fragments are cloned into vector PUG6 to obtain the knockout vector PUG6-LR; the knockout vector PUG6-LR is transformed into the host strain Saccharomyces cerevisiae FRD-SK-25, and the engineered Saccharomyces cerevisiae is obtained after screening.

[0010] Preferably, the nucleotide sequence of the vector PUG6 is shown in SEQ ID NO.3.

[0011] The application of the engineered Saccharomyces cerevisiae in the production of S-adenosylmethionine.

[0012] Preferably, the application method includes the following steps: (1) The engineered strain of Saccharomyces cerevisiae was inoculated onto YPD solid medium and cultured to obtain an activated strain; (2) Select a single colony of the activated strain and inoculate it into YPD liquid medium for culture to obtain seed culture; (3) The seed liquid was inoculated into the fermentation medium for fermentation culture to obtain Saccharomyces cerevisiae containing S-adenosylmethionine; after post-treatment, S-adenosylmethionine was obtained.

[0013] Preferably, the fermentation medium in step (3) includes the following components: glucose, yeast extract, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride, ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium chloride, manganese sulfate monohydrate, copper sulfate, biotin, D-calcium pantothenate, vitamins, and defoamer.

[0014] Preferably, the post-processing step in step (3) includes: breaking the cell wall of the Saccharomyces cerevisiae containing S-adenosylmethionine and centrifuging to remove the cells.

[0015] The application of Saccharomyces cerevisiae containing S-adenosylmethionine in anti-aging and skin barrier repair.

[0016] Preferably, the application method includes the following steps: adding water at 65-75℃ to the Saccharomyces cerevisiae cells containing S-adenosylmethionine, so that the wet weight of the cells reaches 25%-30%; then breaking the cell wall by bathing in a water bath at 65-75℃ for 10-15 minutes to obtain the cell wall-broken liquid; centrifuging the cell wall-broken liquid at 1100-1200 rpm for 4-5 minutes to obtain the supernatant containing S-adenosylmethionine.

[0017] The application of S-adenosylmethionine in the preparation of cosmetics and health foods, wherein the health foods have the effect of assisting in antioxidation.

[0018] Beneficial effects: (1) This invention constructs a high-yield S-adenosylmethionine engineered strain of Saccharomyces cerevisiae by knocking out the glycogen branched chain enzyme GLC3 encoding gene in Saccharomyces cerevisiae FRD-SK-25. The S-adenosylmethionine yield of this engineered strain can reach 12.2 g / L, which is about 87.7% higher than that of the original strain of Saccharomyces cerevisiae FRD-SK-25, providing an excellent strain for the industrial production of S-adenosylmethionine.

[0019] (2) This invention further explores the application of the supernatant containing S-adenosylmethionine obtained by fermentation of the engineered yeast in anti-aging and skin barrier repair, which provides a theoretical basis for the application of S-adenosylmethionine in cosmetics, health food and other fields. Attached Figure Description

[0020] Figure 1 This refers to the double enzyme digestion verification results in Example 1; Figure 2 The spectrum of the pUG6-LR vector knockout; Figure 3 The PCR verification results are for positive transformants; Figure 4 HPLC results for SAM production in engineered and original strains of Saccharomyces cerevisiae. Detailed Implementation

[0021] The following description is based on specific embodiments: brewing yeast ( Saccharomyces cerevisiae FRD-SK-25 was deposited on September 22, 2025 at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, accession number: CGMCC No. 35987.

[0022] Example 1: Construction of engineered Saccharomyces cerevisiae (1) Construction of GLC3 gene knockout vector: The gene sequence of glycogen branched-chain enzyme GLC3 is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. Using the genome of *Saccharomyces cerevisiae* FRD-SK-25 as a template, and using GLC3-F-P1 and GLC3-F-P2 as primers, the left homologous arm fragment of GLC3 was amplified by PCR. The nucleotide sequences of GLC3-F-P1 and GLC3-F-P2 are shown in SEQ ID NOs.4-5, respectively. Using the genome of *Saccharomyces cerevisiae* FRD-SK-25 as a template, and using GLC3-R-P3 and GLC3-R-P4 as primers, the right homologous arm fragment of GLC3 was amplified by PCR. The nucleotide sequences of GLC3-R-P3 and GLC3-R-P4 are shown in SEQ ID NOs.6-7, respectively. Using the Novizan C113-ClonExpress-MultiS One Step Cloning Kit, the left and right homologous arms were sequentially ligated to the linearized starting vector pUG6, which had been digested with Sal1 and Sac11, to obtain the ligation product. The nucleotide sequence of pUG6 is shown in SEQ ID NO.3. The ligation product was transformed into *E. coli* DH5α competent cells and evenly spread on LB agar containing 50 μg / mL ampicillin. The cells were incubated overnight at 37°C with the cells inverted. After incubation, four single clones were randomly selected, plasmids were extracted, and double digestion with Sac1 / Xba1 was performed for verification. The results are shown below. Figure 1 As shown, two clear bands of 4344bp and 1399bp appeared in each lane, indicating that the knockout vector pUG6-LR, which successfully connects the left and right homologous arms of GLC3, was obtained. The pUG6-LR spectrum is shown below. Figure 2 As shown.

[0023] (2) Obtaining GLC3 gene knockout strains: Saccharomyces cerevisiae FRD-SK-25 was streaked in four zones on YPD solid medium and then incubated at 30°C. When a single colony reached 2 mm in diameter, it was picked and inoculated into 3 mL of YPD liquid medium and incubated overnight at 30°C. The culture was then transferred to an Erlenmeyer flask containing 50 mL of YPD liquid medium and incubated further until the OD of the culture medium was measured. 600When the concentration is around 1.0, place the Erlenmeyer flask containing the culture medium on ice for 15 min, then centrifuge at 3000g for 5 min at 4℃, and discard the supernatant. Wash the precipitate twice with 50 mL of ice-cold sterile water, then wash the precipitate again with 20 mL of ice-cold solution A from the Zhuangmeng Biotechnology Yeast Electroporation Competent Cell Preparation Kit, and add 0.5 mL of ice-cold solution B to suspend the cells, obtaining competent yeast cells. Transfer 100 μL of competent yeast cells to a new sterile centrifuge tube, add 2 μg of the knockout vector pUG6-LR, mix well, and then electroporate using an electroporator. Immediately after electroporation, add 500 μL of pre-chilled solution B, mix gently, and then spread on YPD solid medium containing 40 μg / mL G418 resistance for culture until transformants grow. Screen the transformants. Positive transformants exhibited a G418 insensitive phenotype. Genomic DNA was extracted from these transformants, and PCR verification was performed using primers YZ-L and YZ-R. The nucleotide sequences of YZ-L and YZ-R are shown in SEQ ID NOs. 8-9, respectively. The PCR verification results are as follows: Figure 3 As shown, lanes 1 and 2 contain positive transformants, and lane 3 contains the original strain FRD-SK-25, which was transformed from... Figure 3 It was found that the PCR amplification product of the positive transformant had a length difference of 200 bp compared with the PCR amplification product of the original strain FRD-SK-25. One of the positive transformants was selected for induction recombination with the resistance marker G418, thereby obtaining a GLC3 gene knockout strain without G418 resistance, i.e., an engineered Saccharomyces cerevisiae.

[0024] The above-mentioned method for inducing recombination is as follows: Positive transformants phenotyped as insensitive to G418 are uniformly spread onto YPD solid medium containing 10 μg / mL doxycycline and cultured at 30°C until single colonies grow. Then, 100 single colonies are randomly selected and transferred to YPD solid medium and cultured at 30°C for 24 h. Subsequently, each single colony is transferred one-to-one to YPD solid medium containing 40 μg / mL G418 resistance and cultured at 30°C for 24 h. Finally, transformants successfully induced by the resistance marker are screened by phenotypic observation; that is, single colonies that cannot grow on YPD solid medium containing G418 resistance but can grow normally on YPD solid medium are considered successful transformants. KanMX The target transformant was successfully induced to recombine.

[0025] Example 2: Fermentation production of S-adenosylmethionine (SAM) using engineered Saccharomyces cerevisiae The engineered Saccharomyces cerevisiae prepared in Example 1 was fermented in a 20L fermenter to produce SAM. The specific operation steps are as follows: (1) Inoculate the engineered Saccharomyces cerevisiae onto YPD solid medium and incubate it upside down in a 30℃ incubator for 1 day until the cells cover the medium to obtain an activated strain; (2) Select a single colony of the activated strain and inoculate it into YPD liquid medium. Incubate at 29℃ and 220rpm for 16h to obtain seed culture. (3) Transfer 7 mL of seed culture to 900 mL of primary culture medium (YPD liquid culture medium) and culture at 30 °C and 220 rpm for 16 h to obtain primary seed culture; (4) According to the inoculation amount of 20% by volume, 1.8L of primary seed liquid was transferred to a 20L fermenter containing 9L of fermentation medium and fermented at 28℃, 800rpm and aeration rate of 30L / min for 55h to obtain fermentation liquid. The fermentation yeast cells in the fermentation liquid contain SAM. The fermentation broth consists of the following components: glucose 10 g / L, yeast extract 5 g / L, ammonium sulfate 3 g / L, potassium dihydrogen phosphate 8 g / L, magnesium sulfate heptahydrate 1.8 g / L, calcium chloride 0.5 g / L, ferrous sulfate heptahydrate 0.3 g / L, zinc sulfate heptahydrate 0.2 g / L, sodium chloride 0.5 g / L, manganese sulfate monohydrate 0.1 g / L, copper sulfate 0.5 mg / L, biotin 0.1 mg / L, D-calcium pantothenate 3 mg / L, vitamin B 6 mg / L, defoamer 0.01%, and water as the solvent. The pH is adjusted to 5.5, and the mixture is autoclaved at 115°C for 30 minutes.

[0026] Saccharomyces cerevisiae fermentation cells were collected to prepare test samples, and the SAM content in the samples was then determined by high performance liquid chromatography (HPLC). The specific operation steps are as follows: Take 1 mL of fermentation broth, centrifuge at 8000 rpm for 5 min to collect bacterial cells. Wash the bacterial cells once with pure water, then centrifuge again at 8000 rpm for 5 min to collect the bacterial cells. Extract the collected bacterial cells with 1.5 mol / L HClO4 for 1 h, dilute appropriately, filter through a 0.22 μm inorganic filter membrane, and store in a liquid chromatography vial for HPLC detection. The HPLC detection method for SAM is as follows: Agilent high-performance liquid chromatograph with differential detector, C18 analytical column (250 mm × 4.6 mm), mobile phase: ammonium formate (6.3 g ammonium formate, 1.4 g sodium heptanesulfonate, dissolved in 700 mL water, pH adjusted to 3.0 with formic acid, diluted to 1000 mL with water, and shaken well) - methanol (80:20); flow rate 1 mL / min, column temperature 30 ℃, wavelength 260 nm, injection volume 20 μL.

[0027] HPLC detection results are as follows Figure 4 As shown. By Figure 4 As can be seen, compared with the original strain of *Saccharomyces cerevisiae* FRD-SK-25 (original strain), the SAM yield of the engineered *Saccharomyces cerevisiae* strain (recombinant strain) prepared in Example 1 was significantly improved. Specifically, the SAM yield of the original strain was 6.5 g / L, and the SAM yield of the recombinant strain was 12.2 g / L, representing an increase of approximately 87.7%.

[0028] Example 3: Experiment on SAM-regulated expression of antioxidant-related genes after oxidative damage (1) Preparation of supernatant containing SAM: Saccharomyces cerevisiae fermentation cells in the fermentation broth of Example 2 were collected using high-speed refrigerated centrifugation. The cells were washed twice with pure water, and then 70°C pure water was added to achieve a wet weight of 25%. The cells were then subjected to a 70°C water bath for 10 minutes to disrupt the cell walls. The disrupted broth was centrifuged at 1200 rpm for 5 minutes to obtain a supernatant containing SAM (hereinafter referred to as SAM supernatant) for experimental use. The obtained SAM supernatant contained not only SAM but also some secondary metabolites of SAM (see reference for details). Figure 4 ) and yeast lysates.

[0029] (2) H2O2-induced oxidative damage: Human foreskin fibroblasts were digested with HFF and then administered at 0.5 mL / well (each well containing 2 × 10⁶ cells). 5 Cells were seeded into 24-well plates and incubated overnight at 37°C with 5% CO2. Then, oxidative damage was induced in each well by adding 200 μM H2O2, and the plates were incubated at 37°C for 1 hour. Next, SAM supernatant was added to each well at a volume ratio of 5%, while the control group received an equal volume of PBS buffer. Each group was divided into three replicates, and all cells were incubated overnight at 37°C.

[0030] (3) qPCR (real-time quantitative PCR) detection: After culture, the culture medium was discarded, and lysis buffer was added to each well to extract total RNA. RNA concentration and purity were measured, and then reverse transcribed into cDNA. Using GAPDH as an internal reference gene, qPCR was used to detect extracellular matrix-related genes. LN , MKX , COL13A1 Antioxidant-related genes PTEN , SIRT-1 Genes related to the degradation of extracellular matrix MMP Family gene expression. Using a control group gene relative expression fold of F=1, 2... -ΔΔCT The F-values ​​of each sample were calculated using the method described in Table 1.

[0031] Table 1. Expression of relevant genes

[0032] As shown in Table 1, the SAM supernatant prepared in this invention can upregulate extracellular matrix-related laminin. LN Mohawk protein MKX Collagen-like membrane protein 13α chain COL13A1 Antioxidant-related chromosome 10 deletion phosphatases and tensin homologs PTEN Sirtuins protein family 1 SIRT-1 The expression levels of these genes were upregulated by 1.08–3.62-fold; simultaneously, SAM supernatant downregulated the expression of matrix metalloproteinases, a family of proteins associated with the degradation of the extracellular matrix. MMP1 , MMP3 , MMP7 , MMP8 The gene expression levels were downregulated by 0.32–0.95-fold. These results indicate that SAM has a certain anti-aging effect.

[0033] Example 4: Determination of the free radical scavenging ability of SAM supernatant (1) DPPH free radical scavenging ability test The SAM supernatant prepared in Example 3 was tested using the Solarbio DPPH radical scavenging assay kit. Blank, control, and assay groups were set up according to the kit instructions, with three replicates for each group. The OD of each sample was measured. 515nm (A) The DPPH radical scavenging rate of SAM supernatant was calculated. The calculation process and results are shown in Table 2.

[0034] Table 2. Determination of DPPH free radical scavenging ability

[0035] As shown in Table 2, the SAM supernatant prepared in Example 3 has the effect of scavenging DPPH free radicals, with a scavenging rate of 79.12%~84.09%.

[0036] (2) Detection of hydroxyl radical scavenging ability The SAM supernatant prepared in Example 3 was measured using the Solarbio Hydroxyl Radical Scavenging Ability Assay Kit. Blank, control, and assay groups were set up according to the kit instructions, with three replicates for each group. The OD of each sample was measured. 536nm (A) The hydroxyl radical scavenging rate of SAM supernatant was calculated. The calculation process and results are shown in Table 3.

[0037] Table 3. Determination of hydroxyl radical scavenging ability

[0038] As shown in Table 3, the SAM supernatant prepared in Example 3 has the effect of scavenging hydroxyl radicals, with a scavenging rate of 71.43%~78.95%.

[0039] (3) ABTS free radical scavenging ability test The SAM supernatant prepared in Example 3 was tested using the Solarbio ABTS radical scavenging assay kit. Blank, control, and assay groups were set up according to the kit instructions, with three replicates for each group. The OD of each sample was measured. 405nm (A) The ABTS radical scavenging rate of SAM supernatant was calculated. The calculation process and results are shown in Table 4.

[0040] Table 4. Determination of ABTS free radical scavenging ability

[0041] As shown in Table 4, the SAM supernatant prepared in Example 3 has the effect of scavenging ABTS free radicals, with a scavenging rate of 62.67%~68.35%.

[0042] Example 5: Determination of total antioxidant capacity of SAM supernatant The SAM supernatant prepared in Example 3 was measured using the Solarbio Total Antioxidant Capacity (T-AOC) assay kit. Blank and assay groups were set up according to the kit instructions, with three replicates for each group. The OD of each sample was measured. 593nm (A) The total antioxidant capacity of SAM supernatant was calculated. The calculation process and results are shown in Table 5.

[0043] Table 5. Determination of total antioxidant capacity

[0044] As shown in Table 5, the SAM supernatant prepared in Example 3 has antioxidant activity, with a total antioxidant capacity of 2.090~2.180 μmol / mL.

[0045] Example 6: Determination of the skin barrier repairing ability of SAM supernatant Immortalized human keratinocytes (HaCaT) were inoculated into 6-well plates (2 mL / well, 5 × 10⁶ cells per well). 5 Cells were cultured overnight at 37°C in a 5% CO2 incubator until adherence. SAM supernatant was added to each well at a volume ratio of 5%, while the control group received an equal volume of PBS buffer. Each group was divided into triplicate. After 24 hours of culture, the culture medium was discarded, and lysis buffer was added to each well to extract total RNA. RNA concentration and purity were measured, and the RNA was reverse transcribed into cDNA. Using GAPDH as an internal reference gene, qPCR was used for detection. FLG , IVL and OVOL1 Relative gene expression. Using the control group's relative gene expression fold increase (F=1), 2... -ΔΔCT The F-value for each sample is calculated using the following formula: F=2 -ΔΔCT,in: △CT 实验 =CT 实验 -CT 内参（实验） ; △CT 对照 =CT 对照 -CT 内参（对照） ; △△CT=△CT 实验 -△CT 对照 .

[0046] The results are shown in Table 6.

[0047] Table 6. Measurement of Skin Barrier Repair Capacity

[0048] As shown in Table 6, the SAM supernatant prepared in Example 3 can upregulate the filaggrin gene, a skin barrier repair-related factor. FLG outer skin protein gene IVL and OVO-like transcription factor 1 gene OVOL1 The gene expression level was upregulated by 1.21 to 2.27 times, indicating that SAM has the ability to repair the skin barrier.

Claims

1. An engineered strain of brewing yeast, characterized in that, It is brewing yeast ( Saccharomyces cerevisiae The glycogen branched chain enzyme GLC3 encoding gene was knocked out in FRD-SK-25. The Saccharomyces cerevisiae FRD-SK-25 was deposited on September 22, 2025 at the China General Microbiological Culture Collection Center, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, accession number: CGMCC No. 35987.

2. The engineered Saccharomyces cerevisiae strain of claim 1, wherein, The amino acid sequence of the glycogen branched chain enzyme GLC3 is shown in SEQ ID NO.

2.

3. The method for constructing the engineered Saccharomyces cerevisiae strain of claim 1, wherein, The process includes the following steps: using the genome of Saccharomyces cerevisiae strain FRD-SK-25 as a template, the left and right homologous arm fragments of glycogen branched chain enzyme GLC3 are amplified by PCR reaction, and the left and right homologous arm fragments are cloned into vector PUG6 to obtain the knockout vector PUG6-LR; the knockout vector PUG6-LR is transformed into the host strain Saccharomyces cerevisiae FRD-SK-25 to obtain engineered Saccharomyces cerevisiae.

4. The application of the engineered Saccharomyces cerevisiae according to claim 1 in the production of S-adenosylmethionine.

5. The use according to claim 4, wherein the compound is ###0002### The application method includes the following steps: (1) The engineered strain of Saccharomyces cerevisiae was inoculated onto YPD solid medium and cultured to obtain an activated strain; (2) Select a single colony of the activated strain and inoculate it into YPD liquid medium for culture to obtain seed culture; (3) The seed liquid was inoculated into the fermentation medium for fermentation culture to obtain Saccharomyces cerevisiae containing S-adenosylmethionine; after post-treatment, S-adenosylmethionine was obtained.

6. The use according to claim 5, wherein the compound is ###0002### The fermentation medium described in step (3) includes the following components: glucose, yeast extract, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride, ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium chloride, manganese sulfate monohydrate, copper sulfate, biotin, D-calcium pantothenate, vitamins, and defoamer.

7. The use according to claim 5, wherein the compound is ###0002### The post-processing steps in step (3) include: breaking the cell walls of the Saccharomyces cerevisiae containing S-adenosylmethionine and centrifuging to remove the cells.

8. The application of the Saccharomyces cerevisiae containing S-adenosylmethionine as described in claim 5 in anti-aging and skin barrier repair.

9. Use according to claim 8, wherein the compound is ###0002### The application method includes the following steps: adding water at 65-75℃ to the Saccharomyces cerevisiae cells containing S-adenosylmethionine, so that the wet weight of the cells reaches 25%-30%; then breaking the cell wall by bathing in a water bath at 65-75℃ for 10-15 minutes to obtain the cell wall-broken liquid; centrifuging the cell wall-broken liquid at 1100-1200 rpm for 4-5 minutes to obtain the supernatant containing S-adenosylmethionine.

10. The application of S-adenosylmethionine, characterized in that, It is used in the preparation of cosmetics and health foods, wherein the health foods have the effect of assisting in antioxidation.

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