Recombinant saccharomyces cerevisiae with high squalene production and construction method and application thereof

Abstract

The present application relates to the field of synthetic biology and fermentation engineering, and particularly relates to a recombinant Saccharomyces cerevisiae with high squalene production, a construction method and application thereof. The recombinant Saccharomyces cerevisiae is based on Saccharomyces cerevisiae YOS1, squalene synthase gene ERG9 is overexpressed, and squalene epoxidase gene ERG1 is weakly expressed, so as to promote the accumulation of squalene in the cell. Under the optimized fermentation conditions of the general medium, the squalene yield can reach 3062.2 mg / L. Further, based on the results of transcriptome analysis, IDI1, ADH1 and ERG8 genes are expressed in the recombinant Saccharomyces cerevisiae, which can utilize yellow water, a by-product of baijiu, as the main culture medium for squalene fermentation production. The squalene yield can reach 744.4 mg / L, and the chemical oxygen demand removal rate can reach 58.33%. The present application realizes the green and efficient biological manufacturing of squalene and the high-value utilization of yellow water, a by-product of baijiu, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the fields of synthetic biology and fermentation engineering, specifically to a recombinant brewer's yeast that produces high levels of squalene, its construction method, and its applications. Background Technology

[0002] Squalene (C 30 H 50 Squalene, also known as all-trans straight-chain unsaturated olefin, is a natural triterpenoid compound with various biological activities, including antioxidant, anti-inflammatory, and immunomodulatory effects. It is widely used in pharmaceuticals, cosmetics, and functional foods. The main routes for obtaining and synthesizing squalene include three aspects: natural source extraction, chemical synthesis, and biosynthesis. Natural source extraction mainly refers to extraction from deep-sea shark liver or plants (such as olive oil), which suffers from resource limitations, high costs, and ecological damage. Chemical synthesis methods are demanding, have low yields, and cause significant pollution. Although microorganisms cannot naturally accumulate large quantities of squalene like sharks and plants, their rapid growth, convenient cultivation, and mature genetic manipulation make biosynthesis an important candidate system for squalene biomanufacturing. Therefore, developing green and efficient biosynthetic pathways has become a research hotspot.

[0003] brewing yeast ( Saccharomyces cerevisiae Wild-type Saccharomyces cerevisiae possesses advantages such as a clear genetic background, high safety, and mature fermentation technology, making it an ideal host for squalene synthesis. However, squalene, as an intermediate metabolite in ergosterol synthesis, accumulates in extremely low amounts in wild-type Saccharomyces cerevisiae. Current technologies mainly aim to increase yield by overexpressing key enzymes in the mevalonate pathway (such as tHMG1) or weakening squalene epoxidase ERG1, but these methods still suffer from insufficient yield, high fermentation costs, and difficulty in utilizing inexpensive waste materials.

[0004] The brewing process of Baijiu (Chinese liquor) generates a large amount of yellow water, a byproduct with high total acid content, chemical oxygen demand (COD), and biochemical oxygen demand (BOD). Specifically, COD ranges from 250 to 400 mg / mL, and BOD5 from 150 to 300 mg / mL, directly polluting the environment when discharged. While yellow water contains nutrients such as sugars, organic acids, and amino acids, it also contains inhibitors such as phenols and sulfides, limiting its direct use as a microbial culture medium. Therefore, developing a recombinant brewing yeast capable of efficiently producing squalene and adapting to and utilizing inexpensive substrates such as yellow water, while simultaneously exploring the functional applications of its fermentation products, has significant economic and environmental value. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to provide a recombinant brewing yeast that can efficiently produce squalene and adapt to and utilize inexpensive substrates such as yellow water, thereby obtaining functional yeast protein products containing squalene, realizing pollution reduction, energy saving and high-value utilization of yellow water, a by-product of liquor production, and providing a high-squalene-producing recombinant brewing yeast, its construction method and application.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, there is a recombinant brewer's yeast that produces high levels of squalene, wherein the recombinant brewer's yeast is based on brewer's yeast YOS1, and the squalene synthase ERG9 gene of brewer's yeast YOS1 is overexpressed, and the squalene epoxidase ERG1 gene is weakly expressed. The integration site of the squalene synthase ERG9 gene is the 1021b site of the genome of the Saccharomyces cerevisiae YOS1. The specific method for weak expression of the squalene epoxidase ERG1 gene is as follows: the promoter of the squalene epoxidase ERG1 gene of the Saccharomyces cerevisiae YOS1 is replaced with a weak promoter. The weak promoter is the REV1 gene promoter; The sequence of the squalene synthase ERG9 gene is shown in SEQ ID NO: 51; the sequence of the squalene epoxidase ERG1 gene is shown in SEQ ID NO: 52. The brewing yeast YOS1 is erg9::KanMX / CTR3p-ERG9; leu2-3_112::His3MX6 / GAL1p-ERG19 / GAL10p-ERG8;ura3-52::GAL1p-EfMvaS(A110G)- The engineered Saccharomyces cerevisiae strain CYC1t / GAL10p-EfMvaE-ADH1t; his3Δ1::hphMX4 / GAL1p-ERG12 / GAL10p-IDI1;1014a::GAL1p-Erg20.

[0007] The specific construction method of the brewing yeast YOS1 is detailed in the third paragraph on page four of the following document: Pan Luo,Jian-Ming Lv,Yan-Feng Xie, et al. Discovery and characterization of a novel sub-group of UbiA-type terpene cyclases with a distinct motif I. Org. Chem. Front., 2022,9, 3057-3060. Based on the above technical solution, the present invention can be further improved as follows.

[0008] Furthermore, the recombinant Saccharomyces cerevisiae also overexpresses any one of the following genes: mevalonate phosphate kinase ERG8 gene, alcohol dehydrogenase ADH1 gene, fructose-1,6-bisphosphatase FBP1 gene, isoprenyl pyrophosphate isomerase IDI1 gene, and endoplasmic reticulum membrane protein complex subunit OCA1 gene. The sequence of the mevalonate phosphate kinase ERG8 gene is shown in SEQ ID NO: 53; the sequence of the alcohol dehydrogenase ADH1 gene is shown in SEQ ID NO: 54; the sequence of the fructose-1,6-bisphosphatase FBP1 gene is shown in SEQ ID NO: 55; the sequence of the isoprenyl pyrophosphate isomerase IDI1 gene is shown in SEQ ID NO: 56; and the sequence of the endoplasmic reticulum membrane protein complex subunit OCA1 gene is shown in SEQ ID NO: 57.

[0009] Furthermore, the recombinant Saccharomyces cerevisiae also overexpresses the alcohol dehydrogenase ADH1 gene and the isoprenyl pyrophosphate isomerase IDI1 gene; The recombinant Saccharomyces cerevisiae also overexpresses the alcohol dehydrogenase ADH1 gene, the isoprenyl pyrophosphate isomerase IDI1 gene, and the mevalonate phosphate kinase ERG8 gene.

[0010] Furthermore, the site at which the mevalonate phosphate kinase ERG8 gene integrates into the genome of the Saccharomyces cerevisiae YOS1 is int18; The site at which the alcohol dehydrogenase ADH1 gene integrates into the genome of the Saccharomyces cerevisiae YOS1 is int6; The site at which the fructose-1,6-bisphosphatase FBP1 gene integrates into the genome of the Saccharomyces cerevisiae YOS1 is int6; The site at which the isoprenyl pyrophosphate isomerase IDI1 gene is integrated into the genome of the Saccharomyces cerevisiae YOS1 is int4; The site at which the endoplasmic reticulum membrane protein complex subunit OCA1 gene integrates into the genome of the Saccharomyces cerevisiae YOS1 is int6.

[0011] Secondly, a method for constructing a high-squalene-producing recombinant Saccharomyces cerevisiae includes the following steps: using CRISPR / Cas9 technology to integrate the expression cassette of the corresponding overexpressed gene into the genome of the Saccharomyces cerevisiae YOS1, and replacing the natural promoter of the squalene epoxidase ERG1 gene with a weak promoter.

[0012] Thirdly, a method for producing squalene includes the following steps: culturing the high-squalene-producing recombinant Saccharomyces cerevisiae on a culture medium and collecting the squalene from the fermentation products; The culture medium includes YPG medium or pretreated yellow water culture medium, a byproduct of baijiu brewing.

[0013] The pretreatment of the yellow water, a byproduct of Baijiu brewing, includes: adding chitosan and activated carbon to the yellow water for adsorption treatment, filtering to remove solids, adjusting the pH to 6.0, and then centrifuging and microporous membrane filtration.

[0014] Fourthly, a method for the resource utilization of yellow water, a by-product of baijiu brewing, includes the following steps: pre-treating the yellow water and using it as a culture medium to cultivate the high-squalene-producing recombinant brewing yeast, thereby producing squalene while reducing the chemical oxygen demand of the yellow water.

[0015] Fifthly, the application of the high-squalene-producing recombinant Saccharomyces cerevisiae, or the fermentation product prepared by the method for producing squalene, in the preparation of products for relieving intestinal inflammation, improving intestinal flora, or assisting in the relief of colitis. Wherein, the product is a drug, functional food, or feed additive; the fermentation product is Saccharomyces cerevisiae cell fragments or an extract containing squalene.

[0016] This invention focuses on the construction, fermentation optimization, and functional evaluation of high-squalene-producing Saccharomyces cerevisiae in mice with colitis. The invention systematically completed strain modification, process optimization, utilization of yellow water resources, and in vivo activity verification, forming a relatively complete research system.

[0017] The beneficial effects of this invention are: (1) Using Saccharomyces cerevisiae YOS1 as the starting strain, the high-yield squalene engineered strain YOS5 was successfully constructed by enhancing ERG9 expression and weakening ERG1 expression using CRISPR / Cas9 technology. The suitable fermentation process of engineered strain YOS5 under ordinary culture conditions was determined, namely, galactose concentration of 80 g / L, yeast extract to peptone ratio of 1:1, total nitrogen source concentration of 40 g / L, MnSO4 concentration of 0.2 g / L, and CaCl2 concentration of 0.2 g / L. Under these conditions, the squalene yield of YOS5 reached 3062.2 mg / L, which was 9.6 times higher than that of the starting strain. The experimental results show that by enhancing the synthesis throughput and reducing downstream consumption, the accumulation of squalene in Saccharomyces cerevisiae can be effectively promoted.

[0018] (2) The high-squalene-producing Saccharin-producing Saccharin YOS5 product had a good alleviating effect on DSS-induced colitis in C57BL / 6 mice. Animal experiments showed that the fermentation product could improve typical inflammatory phenotypes in mice, such as weight loss, increased spleen index, shortened colon, and colonic tissue damage, while increasing the content of short-chain fatty acids in feces. The non-targeted metabolomics results further showed that after intervention with the high-squalene-producing Saccharin YOS5 product, the overall metabolic profile of mice was significantly improved compared with the model group and was closer to that of the normal group, suggesting that it has a positive effect on alleviating inflammatory response and regulating intestinal metabolic disorders.

[0019] (3) To address the problem of the complex composition of yellow water and the limited ability of conventional engineered strains to produce squalene in it, this invention combines transcriptomics analysis to screen key genes related to yellow water adaptation and product accumulation. Furthermore, genes such as IDI1, ERG8, and ADH1 were introduced into YOS5 to construct an engineered strain, YOS23, suitable for yellow water fermentation. The optimal fermentation conditions for YOS23 in the yellow water system were determined to be: yellow water concentration 80%, galactose addition 80 g / L, pH 6, rotation speed 220 r / min, and temperature 30℃. Under these conditions, the squalene yield of YOS23 in the yellow water medium was approximately 1.7 times higher than that of YOS5, and the fermentation process also showed a certain reduction effect on COD in the yellow water. This indicates that rational modification guided by transcriptomics can effectively improve the adaptability of engineered strains to complex byproduct media and achieve more efficient squalene production under yellow water conditions.

[0020] (4) The high-squalene-producing Saccharin-producing Saccharin YOS23 product obtained by pre-treated yellow water fermentation can also effectively alleviate DSS-induced colitis in mice. Compared with the DSS control group, the YOS23 fermentation product can reduce weight loss and colon damage in mice. The 16S intestinal flora analysis results show that this treatment can promote the restoration of the intestinal flora structure to a normal state, showing a restorative effect on beneficial flora at both the phylum and genus levels, and the overall effect is better than that of the yellow water common Saccharin group. The non-targeted metabolomics results show that it has a significant regulatory effect on related pathways such as amino acid metabolism, lipid metabolism, ABC transporter and tryptophan metabolism, indicating that it may exert its anti-inflammatory effect by synergistically regulating the composition of intestinal flora and host metabolic function. Attached Figure Description

[0021] Figure 1 Figure showing the squalene concentration and dry weight of the squalene-engineered strain; Figure 2 The graph shows the effect of inoculum size on squalene yield. Figure 3 The graph shows the optimization of carbon source fermentation conditions; (A) curves showing the change in squalene yield over time for different carbon sources; (B) squalene yield over 96 hours for different carbon sources. Figure 4 Optimization of fermentation based on carbon source concentration; (A) growth curves at different galactose concentrations and (B) squalene yield at different galactose concentrations over 96 hours; Figure 5 Figure showing the effect of nitrogen source ratio on squalene production by Saccharin in Saccharin; Figure 6 The graph shows the effect of different nitrogen source concentrations on squalene production. Figure 7 This is a graph showing the effect of different salt ions on squalene yield in this invention; Figure 8 The effect of different concentrations of manganese sulfate and calcium chloride on squalene yield is presented in this invention; wherein, (A) manganese sulfate, (B) calcium chloride; Figure 9 The graph shows the change in squalene production of strain YOS5 over time in YP4G and YWM media. Figure 10 GO functional classification diagram of engineered bacteria fermented on YPG and yellow water culture media; Figure 11 Figure showing the fermentation validation results for overexpression of genes related to yellow water adaptation; Figure 12 Figure showing the fermentation validation results for overexpressing genes related to squalene synthesis; Figure 13 The image shows the fermentation validation results for overexpressing genes related to bacterial growth. Figure 14 The fermentation verification diagram shows the recombinant engineered strains; (A) strain YOS20; (B) strain YOS21; (C) strain YOS22. Figure 15 A graph showing the squalene concentration in recombinant strain YOS23; Figure 16 A graph showing the change in squalene production by IEA engineered bacteria over time. Figure 17 Figure 1 shows the effect of yellow water concentration on squalene production by YOS23 engineered bacteria. Figure 18 The effect of galactose concentration on squalene production by YOS23 engineered bacteria is shown in the figure. Figure 19 The graph shows the effect of pH on squalene production by YOS23 engineered bacteria; Figure 20 The graph shows the effect of rotational speed on the squalene yield of YOS23 engineered bacteria; Figure 21 The graph shows the changes in disease activity index and body weight of mice in different treatment groups; (A) daily weight changes of mice during DSS modeling; (B) DAI score. Figure 22Images of the spleen in mice from the NC, DSS, REC, and FPP groups are shown. (A) Image of mouse spleen; (B) Spleen index. Figure 23 Colon length diagrams for mice in the NC, DSS, REC, and FPP groups; Figure 24 PAS staining images of the colon of mice in the NC, DSS, REC, and FPP groups; Figure 25 A graph comparing serum oxidative stress-related indicators in mice from the NC, DSS, REC, and FPP groups, showing colonic inflammatory factors; (A) T-SOD; (B) MDA; (C) GSH. Figure 26 A comparative graph showing the serum inflammatory factor levels of mice in the NC, DSS, REC, and FPP groups; where (A) IL-1β; (B) TNF-α; and (C) CRP. Figure 27 A comparison chart of short-chain fatty acid content in the feces of mice in the NC, DSS, REC, and FPP groups; where (A) acetic acid content; (B) propionic acid content; and (C) butyric acid content. Figure 28 The graphs show the disease activity index and body weight changes of mice in different treatment groups (KB, DSS, HG, HF, and YY); (A) daily body weight changes of mice during DSS modeling; (B) DAI score. Figure 29 Images of the spleen in mice under different treatment groups (KB, DSS, HG, HF, and YY); (A) Image of mouse spleen; (B) Spleen index; Figure 30 Colon length diagrams of mice in different treatment groups (KB, DSS, HG, HF, and YY); Figure 31 A comparison of short-chain fatty acid content in the feces of mice in different treatment groups (KB, DSS, HG, HF, and YY); where (A) acetic acid content; (B) propionic acid content; and (C) butyric acid content. Figure 32 Venn plot showing the shared and unique distribution of OTU / ASV in the gut microbiota of mice in different treatment groups (KB, DSS, HG, HF, and YY); Figure 33 Figure showing the effect of brewer's yeast fermented in yellow water on the phylum level of intestinal flora in mice with colitis; Figure 34The diagram shows the effect of yellow water fermented Saccharomyces cerevisiae on the genus level of intestinal flora in mice with colitis. Among them, (A) columnar stacking of intestinal flora genus; (B) relative abundance of Akkermania genus; (C) relative abundance of Bacteroides genus; (D) relative abundance of Rumenococcus genus; (E) relative abundance of Bacillus genus; (F) relative abundance of Osmotherium genus; (G) relative abundance of Butyricococcus genus. Detailed Implementation

[0022] The principles and features of this invention are described below. The embodiments given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0023] Example 1. Construction of high-squalene-producing Saccharomyces cerevisiae strains and optimization of fermentation process.

[0024] 1.1 Experimental materials.

[0025] 1.1.1 Strains and plasmids.

[0026] The starting engineered strain used in this implementation case is *Saccharomyces cerevisiae* YOS1, which serves as the basic host for subsequent squalene synthesis pathway modification. *Saccharomyces cerevisiae* YOS1 was constructed using *Saccharomyces cerevisiae* CEN.PK-1 (derived from BNCC CEN.PK2-1D, catalog number: BNCC361357) as the starting strain. An engineered strain was constructed using a homologous recombination-mediated site-specific integration strategy. Integration fragments containing target gene expression modules and selection markers were constructed, and sequences homologous to corresponding genomic sites were added to both ends of the fragments to achieve site-specific insertion of the target expression cassette. Specifically, the His3MX6 selection marker and the GAL1p-ERG19 / GAL10p-ERG8 bidirectional expression module were first integrated at the leu2-3,112 site to obtain the leu2-3,112::His3MX6 / GAL1p-ERG19 / GAL10p-ERG8 recombinant strain; then, the GAL1p-EfMvaS(A110G)-CYC1t / GAL10p-EfMvaE-ADH1t expression module was integrated at the ura3-52 site to enhance the upstream metabolic flux of the mevalonate pathway; further, the hphMX4 selection marker and the GAL1p-ERG12 / GAL10p-IDI1 expression module were integrated at the his3Δ1 site to enhance the downstream reaction of the mevalonate pathway and the isoprene precursor conversion capacity. Based on this, the ERG9 site was modified by replacing the natural expression regulatory element of ERG9 with the CTR3 promoter and introducing the KanMX selection marker to obtain the erg9::KanMX / CTR3p-ERG9 conformation, thereby reducing the competitive consumption of precursors such as farnesyl pyrophosphate by the sterol synthesis pathway. Finally, the GAL1p-ERG20 expression module was integrated at the 1014a site of the genome to enhance the synthesis capacity of farnesyl pyrophosphate. After multiple rounds of transformation, screening, and validation, the genotype obtained was erg9::KanMX / CTR3p-ERG9; leu2-3_112::His3MX6 / GAL1p-ERG19 / GAL10p-ERG8;ura3-52::GAL1p-EfMvaS(A110G)- An engineered *Saccharomyces cerevisiae* strain, CYC1t / GAL10p-EfMvaE-ADH1t;his3Δ1::hphMX4 / GAL1p-ERG12 / GAL10p-IDI1;1014a::GAL1p-Erg20, was constructed. Candidate transformants were obtained through screening for corresponding auxotrophic factors or resistance, and each integration site was verified using colony PCR and sequencing. For detailed construction methods, please refer to the following literature: Pan Luo,Jian-Ming Lv,Yan-Feng Xie, et al. Discovery and characterization of a novel sub-group of UbiA-type terpene cyclases with a distinct motif I. Org. Chem. Front., 2022,9, 3057-3060.The ERG12 sequence is shown in SEQ ID NO: 58; the ERG20 sequence is shown in SEQ ID NO: 59; the ERG19 sequence is shown in SEQ ID NO: 60; the MvaS(A110G) sequence is shown in SEQ ID NO: 61; the MvaE sequence is shown in SEQ ID NO: 62; the pCTR3 sequence is shown in SEQ ID NO: 63; and the KanmX sequence is shown in SEQ ID NO: 64. The plasmid strain is Escherichia coli. E. coli -TOP10, used for the construction, amplification, and preservation of target plasmids; among them, pESC-Ura is a yeast shuttle expression plasmid used for promoter and terminator fusion of target genes and preparation of gene expression cassettes; pCut-1021b is a CRISPR / Cas9 gene editing plasmid carrying a Cas9 expression cassette and sgRNA expression elements, used for specific cleavage of the 1021b site in Saccharomyces cerevisiae. The strains and plasmids are shown in Tables 1 and 2.

[0027] Table 1. Gene information of strains and plasmids constructed by overexpressing ERG9 and weakening ERG1. Table 2. Strains and plasmid gene information of high-squalene-producing Saccharin-producing Saccharin-producing yeast based on yellow water utilization. 1.1.2 Main reagents.

[0028] Molecular biology reagents 2× Taq PCR Master Mix and 2× HiFiMax Master Mix were purchased from Nanjing Novizan Biotechnology Co., Ltd. SanPrep column DNA gel extraction kit and SanPrep column plasmid DNA mini-extraction kit were purchased from Shanghai Sangon Biotech Co., Ltd.

[0029] 1.1.3 Culture medium.

[0030] ① LB medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl. Sterilize at 115℃ for 20 min, then add appropriate antibiotics as needed. ② YPD medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, natural pH. Sterilize at 115℃ for 20 min. ③ YPG medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L galactose, natural pH. Sterilize at 115℃ for 20 min. ④ SD-Leu medium: 8 g / L Leu, 20 g / L glucose, 20 g / L galactose, 0.17 g / L agar, natural pH. Sterilize at 115℃ for 20 min. ⑤ 100 mg / mL Ampicillin Stock Solution: Weigh 2 g of ampicillin and dissolve it in 20 mL of water. Filter sterilely through a 0.22 μm filter head, sterilize, aliquot, and store at -20℃. Dilute 1000 times before use. ⑥ Yellow Water Culture Medium (YWM): Yellow water was collected from a batch of fermentation water from the Jinpai Fenglin Winery in Yangxin County. First, 2% chitosan (pH 4) was added to the yellow water, followed by 5% 12-mesh activated carbon. Adsorption was performed at 40℃ for 30 min. Filtering was then performed to remove activated carbon and large particulate impurities. The pH of the filtrate was adjusted to 6, and centrifuged at 8000 ×g for 20 min. The supernatant was collected and further filtered using a 0.45 μm microporous membrane. Then, 4% galactose was added to prepare the yellow water culture medium.

[0031] 1.2 Experimental methods.

[0032] 1.2.1 Brewing yeast culture method.

[0033] Saccharomyces cerevisiae strains preserved at -80℃ were streaked onto YPD solid plates and incubated upside down at 30℃ for 48 h. After single colonies with regular morphology and clear edges had grown, they were selected for further experiments.

[0034] Saccharomyces cerevisiae were selected from YPD culture dishes and inoculated into 50 mL shake flasks containing 10 mL of YPD liquid medium. The cultures were incubated at 30℃ and 220 r / min for 18 h with shaking to obtain primary seed culture. The primary seed culture was then transferred at an inoculation rate of 16% (v / v) to 250 mL Erlenmeyer flasks containing 50 mL of YPG liquid medium and incubated at 30℃ and 220 r / min for 96 h with shaking. Samples were taken at 24 h, 48 h, 72 h, and 96 h to determine squalene yield.

[0035] 1.2.2 Squalene extraction method.

[0036] Liquid nitrogen grinding extraction method: Pre-cool the mortar in an ultra-low temperature freezer at -80℃ for 30 min; take 5 mL of fermentation broth, centrifuge at 5,000 r / min and 4℃ for 10 min, and collect the bacterial cells; place the bacterial cells in a pre-cooled mortar, add liquid nitrogen for protection, and grind rapidly for 20 min, replenishing liquid nitrogen every 2 min during this period to prevent bacterial cell degradation due to temperature rise; quickly transfer the ground bacterial cell powder to a 2 mL centrifuge tube, add 1 mL of ethyl acetate, and vortex for 30 s to ensure that the sample and the extract are in full contact and complete the extraction; then centrifuge at 4℃ and 12,000 r / min for 10 min, and collect the supernatant for later use.

[0037] The extraction method was performed in triplicate, and the squalene extraction rate was determined by gas chromatography to screen the optimal extraction process. 1.2.3 Detection method for squalene.

[0038] High-performance gas chromatography (GC) was used for qualitative and quantitative detection of squalene. The specific conditions and procedures are as follows: (1) Detection instrument: Agilent 7890B, GC-FID. The chromatographic column was a DB-5 capillary column (30 m × 0.32 mm, 0.25 μm). The injection port temperature was 300℃, the detector temperature was 350℃, and the injection volume was 10 μL. The column temperature program was set as follows: initial temperature 160℃, hold for 5 min; then increase the temperature to 325℃ at 10℃ / min and hold for 8 min, with a total run time of 26.5 min. The carrier gas was nitrogen, the flow rate was 1 mL / min, and the split ratio was 10:1. Squalene was quantified using the external standard method; the specific steps are as follows: Standard curve preparation: using ethyl acetate as solvent, squalene standard solutions with concentration gradients of 10 mg / L, 100 mg / L, 500 mg / L, 1100 mg / L, 3000 mg / L, and 4000 mg / L were prepared; the standard curve was plotted, and the regression equation (R) was calculated. 2 (≥0.999 is considered acceptable); samples obtained by each extraction method were tested under the same conditions, peak areas were recorded, and the peak areas were substituted into the regression equation to calculate the concentration and extraction rate of squalene in the samples.

[0039] 1.2.4 Method for constructing engineered strains that overexpress ERG9 and weaken ERG1.

[0040] Using Saccharomyces cerevisiae YOS1 as the starting strain, ERG9 was overexpressed and ERG1 was attenuated using CRISPR / Cas9 technology.

[0041] 1.2.4.1 Crude extraction of brewer's yeast.

[0042] The yeast genome was rapidly extracted using the alkaline lysis method. The specific steps are as follows: (1) Pick a single colony on a YPD plate, add 20 μL of 20 mM NaOH solution to a 1.5 mL centrifuge tube, and resuspend it by pipetting; (2) Place the centrifuge tube in a 95℃ metal bath for 20 min to lyse the yeast cells and release the genomic DNA; (3) Centrifuge at 10,000 ×g for 1 min at room temperature, and take the supernatant as a PCR amplification template for later use.

[0043] 1.2.4.2 Plasmid extraction.

[0044] Plasmids were extracted from E. coli using the SanPrep column-based plasmid DNA mini-extraction kit, and the procedure was strictly followed according to the kit instructions.

[0045] 1.2.4.3 DNA fragment amplification.

[0046] (1) Low-fidelity enzyme PCR amplification.

[0047] For yeast genome verification, colony PCR, and preliminary plasmid screening, 2× Taq PCR Master Mix was used as the amplification enzyme. The reaction system and procedure are as follows: Reaction system (50 μL): 25 μL 2× Taq PCR Master Mix, 2.5 μL each of forward and reverse primers (10 μM), 2 μL template DNA, and ddH2O to 50 μL; Reaction procedure: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 62℃ annealing for 30 s, 72℃ extension for 1 min / kb, 25~35 cycles; 72℃ complete extension for 10 min; 4℃ incubation.

[0048] (2) High-fidelity PCR amplification.

[0049] For amplification of target gene expression cassettes, plasmid backbones, and homologous arm fragments, 2×HiFiMax Master Mix was used as the amplification enzyme. The reaction system and procedure are as follows: Reaction system (50 μL): 25 μL 2×HiFiMax Master Mix, 2.5 μL each of forward and reverse primers (10 μM), x μL template DNA, ddH2O added to 50 μL; Reaction procedure: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 62℃ annealing for 20 s, 72℃ extension for 30 s / kb, 25~35 cycles; 72℃ complete extension for 10 min; 4℃ incubation.

[0050] 1.2.4.4 Gel recovery of PCR products.

[0051] The target DNA fragment was recovered using the SanPrep column-based DNA gel recovery kit.

[0052] 1.2.4.5 Plasmid construction and validation.

[0053] Based on the principle of homologous recombination, a recombinant plasmid was constructed and ligated using the ClonExpressⅡ One Step Cloning Kit (Novizan). The specific steps are as follows: (1) Fragment preparation: The target gene fragment (with homologous arms) and the plasmid backbone fragment were amplified by high-fidelity PCR, and then purified by gel extraction and quantification; (2) Homologous recombination ligation to obtain the ligation product. (3) E. coli transformation; (4) Positive clone verification; (5) Sequencing verification: sent to Sanger sequencing by Sangon Biotech (Shanghai) Co., Ltd. After the sequence was consistent with the target sequence, it was used for subsequent Saccharomyces cerevisiae transformation.

[0054] 1.2.4.6 Saccharomyces cerevisiae transformation (CRISPR / Cas9 mediated).

[0055] The target gene was inserted into the Saccharomyces cerevisiae genome at a specific site using a PEG / LiAc-mediated chemical transformation method combined with CRISPR / Cas9 gene editing technology. The steps are as follows: (1) Preparation of competent yeast cells: Single clones of Saccharomyces cerevisiae were picked from YPD plates, inoculated into YPD liquid medium, and cultured. The supernatant was discarded, the cells were resuspended in pre-cooled dd H2O, and then washed with LiAc solution to obtain competent yeast cells. (2) Preparation of transformation system: 16 μL of dd H2O, 1 μg of homologous repair fragment (gene expression cassette with homologous arms), 500 ng of Cas9 expression plasmid, and 1 μg of sgRNA expression plasmid were added to the competent yeast cells in sequence. After mixing, the cells were placed on ice. Then 240 μL of PEG3350, 36 μL of 1 M LiAc, and 20 μL of ssDNA (the order of addition cannot be changed) were added and vortexed for 10 s to mix. After induction culture and screening, positive clone screening, and transformant verification, the cells were stored in glycerol at -80℃ for subsequent fermentation experiments.

[0056] 1.2.4.7 Overexpression of squalene synthase (ERG9).

[0057] Using CRISPR / Cas9 gene editing technology, we achieved site-specific overexpression of the squalene synthase gene ERG9 in the genome of Saccharomyces cerevisiae, and investigated the effect of peroxisome-mediated expression on squalene accumulation. The specific steps are as follows: (1) Construction of the ERG9 gene expression cassette.

[0058] Using the Saccharomyces cerevisiae genome as a template, high-fidelity PCR amplification was performed using primers pERG9-F and pERG9-R, and the ERG9 gene fragment containing a 20 bp homologous arm of the pESC-Ura plasmid was obtained by gel extraction; using the pESC-Ura plasmid as a template, high-fidelity PCR amplification was performed using primers pESC-SKE-F and pESC-SKE-R, and the plasmid backbone fragment was obtained by gel extraction. The ERG9 gene fragment was ligated to the plasmid backbone using homologous recombination and transformed into... E. coli After the top 10 competent cells were verified to be correct by colony PCR and sequencing, the recombinant plasmid (pESC-Ura-ERG9) was extracted. Using the recombinant plasmid as a template, high-fidelity PCR amplification was performed using primers p1021b-F and p1021b-R. The ERG9 gene expression cassette (containing the promoter, ERG9 gene, and terminator) was obtained by gel recovery, and the expression cassette had 40 bp homologous arms at both ends of the 1021b site.

[0059] (2) Overexpression of ERG9 gene at site 1021b.

[0060] Using pCut-1021b plasmid as a medium, the above-mentioned ERG9 gene expression cassette was inserted into the 1021b site of the Saccharomyces cerevisiae genome via CRISPR / Cas9-mediated transformation. The ERG9 overexpressing strain was obtained after PCR and sequencing verification.

[0061] The primers used for overexpressing squalene synthase are detailed in Table 3.

[0062] Table 3 Primers used for overexpression of squalene synthase 1.2.4.8 Weakening of the squalene oxygenase (ERG1) promoter.

[0063] By employing a promoter substitution strategy, the expression of the squalene oxygenase gene ERG1 was weakened, thereby reducing squalene degradation. The specific steps are as follows: Construction of pCut-pERG1 plasmid: Using pCut-1021b plasmid as a template, high-fidelity PCR amplification was performed using primers CRISPR-R and pKOpERG1-F, and primers CRISPR-F and pKOpERG1-R, respectively. The two fragments were recovered from the gel, ligated via homologous recombination, and then transformed into pCut-pERG1. E. coli -TOP10, after sequencing verification, the pCut-pERG1 recombinant plasmid was obtained (for site-directed cleavage of the ERG1 promoter). (1) Amplification of weak promoter fragments: Six candidate promoters were selected (constitutive weak promoters REV1, SAC6, ATP3, TDH1; galactose-inducible promoters GAL7, GAL2), among which GAL7 and GAL2 promoters have the characteristics of "high expression in the early stage (beneficial to yeast growth) and low expression in the later stage (beneficial to squalene accumulation)". Using the Saccharomyces cerevisiae genome as a template, specific primers were designed (Table 4), and the six promoter fragments were amplified by high-fidelity PCR. Each promoter fragment has homologous arms at both ends of the ERG1 promoter internal site required for insertion. Table 4. Primers used for promoter replacement (2) Promoter replacement and transformation: Using pCut-pERG1 plasmid as a medium, the above 6 promoter fragments were respectively transformed into Saccharomyces cerevisiae using CRISPR / Cas9-mediated transformation method to cut the ERG1 promoter of Saccharomyces cerevisiae at specific sites and replace the original ERG1 promoter; after screening and verification of positive strains, fermentation verification was carried out. The 6 strains were shake-flask fermented, the squalene yield was measured, and the engineered strain with the best ERG1 attenuation effect was screened for further experiments.

[0064] 1.2.5 Optimization of fermentation process for engineered strains that overexpress ERG9 and weaken ERG1.

[0065] (1) Single-factor experiment on fermentation conditions.

[0066] Using the engineered strain YOS5 as the research object and squalene yield as the evaluation index, the effects of inoculum size, carbon source, nitrogen source, and inorganic salts on fermentation efficiency were investigated. Each factor was set with 5 levels and 3 replicates. The fermentation conditions were fixed as follows: 250 mL shake flask, 50 mL liquid volume, 30℃, 220 r / min, and 96 h fermentation. The single-factor experimental design is as follows: The inoculum sizes were designed to be 2%, 9%, 16%, 23%, and 30%; the carbon source combinations were designed to be 20 g / L fructose + 20 g / L galactose, 20 g / L sucrose + 20 g / L galactose, 20 g / L glycerol + 20 g / L galactose, 20 g / L glucose + 20 g / L galactose, and 40 g / L galactose; the total carbon source concentrations were designed to be 20 g / L, 40 g / L, 60 g / L, 80 g / L, and 100 g / L; the yeast extract:peptone ratios were designed to be 2:1, 1:1, 1:2, 1:3, and 1:4; the total nitrogen source concentrations were designed to be 10 g / L, 20 g / L, 30 g / L, 40 g / L, and 50 g / L; and the inorganic salts were designed to be added at 0.2 g / L each. Different inorganic salts (ZnSO4, MgSO4, CuSO4, CaCl2, MnSO4, NaCl, K2HPO4, CH3COONH4) at g / L.

[0067] The optimal fermentation conditions were determined by comparing the squalene concentration and dry weight under different fermentation conditions.

[0068] (2) Orthogonal experiment of fermentation conditions.

[0069] Orthogonal experimental designs were conducted for nitrogen source concentration, MnSO4 concentration, and CaCl2 concentration, respectively, with squalene yield as the evaluation index, to optimize fermentation process parameters. The specific designs are as follows (Table 5): Table 5. Factors and Levels in the Orthogonal Experiment 1.2.6 Method for constructing a high-squalene-producing Saccharin-producing Saccharin yeast based on yellow water utilization.

[0070] Using the previously constructed high-squalene-producing Saccharomyces cerevisiae YOS5 as the starting strain, key genes related to yellow water adaptability were screened by combining transcriptome analysis, and the strain was then targeted by combined overexpression to construct an engineered strain suitable for yellow water fermentation.

[0071] 1.2.6.1 Preparation of transcriptome sequencing samples.

[0072] The starting strain was inoculated at a 16% inoculum into YP4G conventional synthetic medium and YWM yellow water medium, with three replicates per group, and shake-flask fermentation was performed. During fermentation, samples were taken every 24 hours, and the squalene content at each time point was detected to screen for the time point with the most significant change in squalene content. The samples from this time point were then sent to Sangon Biotech (Shanghai) Co., Ltd. for total RNA extraction, purification, and transcriptome sequencing, laying the foundation for subsequent differential gene screening.

[0073] 1.2.6.2 Screening for the target gene to improve squalene yield through yellow water fermentation.

[0074] The raw data obtained from transcriptome sequencing were filtered, aligned, assembled, and subjected to differential expression analysis. Strains cultured in YPG conventional synthetic medium served as controls, while strains cultured in yellow water medium served as treatment groups. The focus was on screening three key differentially expressed genes to identify the key gene targets that restrict *Saccharomyces cerevisiae*'s adaptation to yellow water environment, efficient growth, and squalene synthesis. The specific screening criteria are as follows: (1) Yellow water adaptation-related genes: compared with the conventional synthetic medium YP4G, the genes that were significantly highly expressed in the yellow water medium YWM were speculated to be involved in the adaptive processes of yeast to the tolerance of inhibitory substances in yellow water and the utilization of nutrients. (2) Genes related to cell growth: Compared with YWM yellow water medium, the genes that are significantly highly expressed in YP4G conventional synthetic medium are speculated to be involved in growth-related processes such as yeast cell proliferation and metabolic homeostasis maintenance. (3) Squalene synthesis-related genes: Compared with YWM yellow water medium, genes that are significantly highly expressed in YP4G conventional synthesis medium are of particular interest. We focus on key genes in the mevalonate (MVA) pathway and squalene synthesis pathway, and speculate that they may directly affect the squalene synthesis efficiency.

[0075] 1.2.6.3 Overexpression of the target gene to increase squalene yield through yellow water fermentation.

[0076] Homologous recombination technology was used to construct Cas9 knock-in plasmids pCut-int4 and pCut-int18 for site-specific knock-in of the target gene. The specific construction steps are as described above, and the primer sequences used are shown in Table 6. Table 6 Primers used for Huangshui gene overexpression The key genes identified through screening were individually overexpressed and validated. The specific procedures were as follows: First, specific primers were designed based on the target gene sequence, and the target gene fragment was amplified using PCR technology. Then, the amplified target gene fragment was ligated into the pESC-URA vector to construct a recombinant expression plasmid. The target gene expression cassette with homologous arms at both ends of the int6 site was obtained through PCR amplification, and the nine key genes identified through screening were transformed into the int6 site of the YOS5 strain. After construction, the squalene yield of each gene-overexpressing strain was detected through shake-flask fermentation experiments. Genes that significantly increased squalene yield were screened for further combined overexpression validation.

[0077] 1.2.6.4 Optimization of fermentation process for high-squalene-producing Saccharin-producing Saccharin yeast based on yellow water utilization.

[0078] (1) Determination of the fermentation time of the target strain in yellow water.

[0079] Squalene production was induced using yellow water as the base culture medium with 4% galactose added as an inducer. The fermentation conditions were set as follows: initial pH = 6, fermentation temperature 30℃, shaking speed 220 r / min, and inoculum size 18%. During fermentation, samples were taken every 12 h to detect the squalene yield in the samples. A curve showing the change in squalene yield over fermentation time was plotted. Based on the curve's variation, the optimal fermentation time for the target strain in yellow water was determined.

[0080] (2) Single-factor fermentation experiment.

[0081] Using the YOS23 engineered strain as the research object, the basic fermentation conditions were fixed as follows: fermentation time 96 h, fermentation temperature 30℃, shaking speed 220 r / min, initial pH 6, yellow water concentration 20%, inoculum size 16%, and galactose addition 4 g / L. The following single factors were varied to investigate their effects on squalene yield. Three replicates were set for each treatment, and the average value was taken as the final result. The specific factors and gradient settings are as follows: The concentrations of the yellow water were designed to be 20%, 40%, 60%, 80%, and 100% (v / v); the initial pH was designed to be the initial pH of the yellow water (3.8), 5, 6, 7, and 8; the galactose concentrations were designed to be 20 g / L, 40 g / L, 60 g / L, 80 g / L, and 100 g / L; and the shaking speeds were designed to be 160 r / min, 190 r / min, 220 r / min, 250 r / min, and 280 r / min.

[0082] (3) Orthogonal experiment.

[0083] Based on the results of single-factor experiments, three factors (yellow water concentration, pH, and rotation speed) that significantly affect squalene yield were selected. An orthogonal experimental design was adopted, using squalene yield (mg / L) as the evaluation index, to further optimize the fermentation process parameters and clarify the influence weight of each factor and the optimal combination of levels. The orthogonal experimental factors and level settings are shown in Table 7, with the blank column (A) used to estimate the experimental systematic error.

[0084] Table 7. Factors and Levels in the Orthogonal Experiment 1.2.7 Statistical analysis.

[0085] All experiments were conducted three times. Analysis of variance (ANOVA) was used to describe the significance of the data, and p < 0.05 was considered statistically significant.

[0086] This invention adopts the national standard HJ 828-2017 "Determination of Chemical Oxygen Demand in Water - Potassium Dichromate Method" to detect and statistically analyze COD.

[0087] 1.3 Results and Analysis.

[0088] 1.3.1 Determination of squalene standard curve.

[0089] A scatter plot was drawn with squalene concentration as the x-axis (X mg / L) and peak area as the y-axis, and linear regression analysis was performed. The corresponding conversion formula between peak area and concentration was obtained as: X = (Y - 6.74) / 1.652. The correlation coefficient R of this regression equation was [missing value]. 2A value of ≥0.999 indicates that the squalene concentration exhibits a good linear relationship with the peak area in the range of 10~4000 mg / L, which can be used for accurate calculation of the squalene concentration in subsequent samples.

[0090] 1.3.2 Effects of overexpression of squalene synthase on bacterial growth and squalene accumulation.

[0091] Using the original strain as a control, overexpression of the squalene synthase gene ERG9 increased squalene production from 266.22 mg / L in YOS1 to 1075.18 mg / L in YOS2, representing a 303.86% increase, approximately 4.04 times, compared to the original strain. This result indicates that enhancing ERG9 expression can significantly increase the allocation of metabolic flux to the squalene synthesis branch, and is an effective strategy for promoting squalene accumulation.

[0092] 1.3.3 Effects of weakening squalene cyclooxygenase expression on bacterial growth and squalene accumulation.

[0093] Six candidate promoters (REV1, SAC6, ATP3, TDH1, GAL7, and GAL2) were selected to construct six recombinant strains with weakened ERG1 promoters. Using *Saccharomyces cerevisiae* YOS2 overexpressing ERG9 as a control group, shake-flask fermentation experiments were conducted to detect squalene production. The results are as follows: Figure 1 As shown.

[0094] Experimental results showed that the squalene production of recombinant strains differed significantly after different promoter replacements (P<0.05): Replacing YOS5 and YOS10 with REV1 and GAL2 promoters significantly increased squalene production, reaching 1415.7 mg / L and 1226.1 mg / L respectively, representing increases of 32.86% and 10.43% compared to the control group YOS2; while the squalene production of strains replaced with SAC6, GAL7, TDH1, and ATP3 promoters was lower than the control group, indicating poor attenuation. Overall, the REV1 promoter attenuation effect was optimal, and YOS5 recombinant strain was selected for further optimization of fermentation conditions.

[0095] 1.3.4 Results of single-factor fermentation conditions for YOS5.

[0096] (1) Effect of inoculum size on squalene yield.

[0097] like Figure 2The results showed that after 96 h of fermentation, the squalene yields under different inoculum sizes were: 1053.3 mg / L (2%), 1116.2 mg / L (9%), 1441.3 mg / L (16%), 1252.3 mg / L (23%), and 1174.3 mg / L (30%). The squalene yield was highest at an inoculum size of 16%, significantly higher than other inoculum sizes (P<0.05).

[0098] (2) The effect of carbon source on the synthesis of squalene by recombinant bacteria.

[0099] like Figure 3 Figures A and B show that squalene yield initially increased and then stabilized with increasing fermentation time, reaching its highest value at 96 h, indicating that 96 h is the optimal fermentation time. At 96 h, there were significant differences in squalene yield among different carbon source combinations (P<0.05): the highest squalene yield (1441.9 mg / L) was achieved with 40 g / L galactose as the sole carbon source; the second highest yield (1360.3 mg / L) was achieved with the combination of 20 g / L sucrose and 20 g / L galactose; and the lowest yield (968.3 mg / L) was achieved with the combination of 20 g / L glycerol and 20 g / L galactose.

[0100] Experimental results showed that galactose, as the sole carbon source, was more beneficial to the growth of strain YOS5 and squalene synthesis, possibly because the presence of other carbon sources was detrimental to the induction of GAL series promoters (squalene synthesis is induced by GAL promoters). Therefore, further experiments selected galactose as the primary carbon source.

[0101] (3) Effect of galactose concentration on squalene production.

[0102] like Figure 4 Figures A and B show that within the concentration range of 20–100 g / L, squalene production initially increased and then decreased with increasing galactose concentration, exhibiting a significant dose-response effect (P<0.05). At a galactose concentration of 80 g / L, squalene production reached its highest value of 2298.3 mg / L, representing a 149.09% increase compared to the 20 g / L group; however, when the galactose concentration continued to increase to 100 g / L, squalene production actually decreased.

[0103] (4) Effect of nitrogen source ratio on squalene yield.

[0104] like Figure 5The results showed that squalene yield varied significantly under different nitrogen source ratios (P<0.05): the highest squalene yield was achieved when the ratio of yeast extract to peptone was 1:1, reaching 2355.5 mg / L; as the peptone ratio increased (ratios of 1:2, 1:3, and 1:4), the squalene yield gradually decreased, with the lowest yield at a ratio of 1:4, reaching only 1677.1 mg / L; the yield was also lower than the optimal group when the yeast extract ratio was too high (ratio of 2:1).

[0105] (5) Effect of nitrogen source concentration on squalene production.

[0106] like Figure 6 The results showed that squalene production first increased and then decreased with the increase of total nitrogen source concentration: when the total nitrogen source concentration was 40 g / L (20 g / L yeast extract and 20 g / L peptone), squalene production reached the highest level of 2414 mg / L.

[0107] (6) The effect of inorganic salts on the synthesis of squalene by recombinant bacteria.

[0108] like Figure 7 The results showed that the addition of 0.2 g / L MgSO4, 0.2 g / L CaCl2, and 0.2 g / L MnSO4 all increased squalene production, reaching 2572.7 mg / L, 2634.1 mg / L, and 2681.5 mg / L, respectively, representing increases of 11.23%, 13.88%, and 15.93% compared to the control group. However, the addition of 0.2 g / L ZnSO4 and 0.2 g / L CuSO4 decreased squalene production, suggesting that these two ions may inhibit the activity of squalene synthase or interfere with the normal metabolism of yeast cells. The addition of CH3COONH4, NaCl, and K2HPO4 did not show a significant difference in production compared to the control group (P>0.05).

[0109] For MnSO4 and CaCl2, which have significant promoting effects, concentration gradients (0 g / L, 0.2 g / L, 0.4 g / L) were set to further optimize their addition concentrations. The results are as follows: Figure 8 As shown in Figures A and B. The results of the concentration optimization experiment indicate that ( Figure 8 In both cases (A and B), the highest squalene yield was achieved when the concentrations of MnSO4 and CaCl2 were both 0.2 g / L, reaching 2681.5 mg / L and 2629.2 mg / L, respectively. When the concentration increased to 0.4 g / L, the yield decreased in both cases, suggesting that excessively high ion concentrations disrupt the homeostasis of the yeast cell environment and inhibit metabolic enzyme activity. Therefore, the optimal addition concentrations of MnSO4 and CaCl2 were determined to be 0.2 g / L.

[0110] 1.3.5 Results of orthogonal experiments on fermentation conditions of strain YOS5 constructed.

[0111] Based on the results of single-factor experiments, orthogonal experiments were conducted to optimize the nitrogen source concentration, MnSO4, and CaCl2, with squalene yield as the indicator. Analysis of the experimental results showed that, within the experimental design range, the optimal experimental scheme was: a total nitrogen source concentration of 40 g / L, a MnSO4 concentration of 0.2 g / L, and a CaCl2 concentration of 0.2 g / L. Analysis of variance showed that the nitrogen source concentration had a significant impact on the yield.

[0112] It was found through single-factor and orthogonal experiments that the optimal concentrations for squalene production were 80 g / L galactose, 1:1 yeast extract to peptone ratio, 40 g / L total nitrogen source, 0.2 g / L MnSO4, and 0.2 g / L CaCl2, resulting in a yield of 3062.2 mg / L, which was 9.6 times higher than the starting strain. This indicates that the constructed strain has a strong squalene synthesis capacity and that optimized fermentation process significantly promotes yield improvement.

[0113] 1.3.6 Determination of transcriptome sequencing sampling time.

[0114] To investigate the regulatory mechanism of yellow water culture medium on squalene synthesis in Saccharomyces cerevisiae, the optimal sampling time for transcriptome sequencing was first determined, and the squalene accumulation of strains at different fermentation time points was dynamically monitored. Based on... Figure 9 As a result, in the YP4G conventional synthesis medium, squalene production increased rapidly from 24 to 48 h, indicating that the cells were growing and metabolizing vigorously during this period, and their squalene synthesis capacity was continuously enhanced. In contrast, in the yellow water medium (YWM), the squalene accumulation rate was significantly lower than that in the YP4G medium, and the difference between the two groups increased significantly from 48 h onwards (P<0.05).

[0115] 1.3.7 Transcriptome data analysis and target gene screening.

[0116] This invention performed high-throughput sequencing on six samples: YPG1, YPG2, YPG3, and YWM1, YWM2, and YWM3. The experimental results showed that the sequencing data for each sample was sufficient, with a high total sequence number ranging from 0.4 to 1.5 G, and a Q30 of 98.1% to 98.2%, indicating good sequencing quality and reliable data that meets the requirements for subsequent transcriptome analysis. Using YOS5 strain cultured in YP4G medium as the control group and YOS5 strain cultured in YWM medium as the treatment group, differential expression analysis of the transcriptome sequencing data revealed 1716 differentially expressed genes, of which 714 genes were significantly upregulated and 1002 genes were significantly downregulated, clarifying the target range for subsequent gene screening and strain modification.

[0117] Based on this, GO functional classification annotations were performed on differentially expressed genes (e.g. Figure 10 GO functions are mainly divided into three categories: biological processes, cellular components, and molecular functions. The results showed that differentially expressed genes were distributed in all three functional categories, with the highest number of entries related to biological processes, indicating that these genes are mainly involved in various metabolic and life activities within the cell. Based on the screening criteria, the differentially expressed genes were functionally annotated and classified, and key genes related to squalene synthesis, bacterial growth, and yellow water adaptation were identified, as follows: (1) Differential genes related to bacterial growth.

[0118] Compared to YWM medium, the three cell growth-related genes with the highest expression levels in YP4G medium for strain YOS5 were ERG12 (encoding gene for mevalonate kinase), GAL1 (encoding gene for galactokinase), and OCA1 (encoding gene for endoplasmic reticulum membrane protein complex (EMC) subunit).

[0119] (2) Differential genes related to squalene synthesis.

[0120] Compared to YWM medium, the squalene synthesis-related genes highly expressed by strain YOS5 in YP4G medium mainly involve the mevalonate (MVA) pathway, including ERG8 (encoding gene for mevalonate phosphate kinase), IDI1 (encoding gene for isoprenyl pyrophosphate isomerase), and MVD1 (encoding gene for mevalonate pyrophosphate decarboxylase) in addition to ERG12.

[0121] (3) Differential genes related to yellow water adaptation.

[0122] Compared to conventional culture media, the YOS5 strain in YWM medium highly expressed yellow water adaptation-related genes mainly include ADH1 (encoding gene for alcohol dehydrogenase), FBP1 (encoding gene for fructose-1,6-bisphosphatase), and FDH1 (encoding gene for formate dehydrogenase).

[0123] Detailed expression information of the key differentially expressed genes obtained from the above screening is shown in Table 8.

[0124] Table 8. Key differentially expressed gene information for screening. 1.3.8 Validation of target gene overexpression.

[0125] Based on the screening and functional annotation of differentially expressed genes in the transcriptome, genes related to yellow water adaptation (ADH1, FBP1, FDH1), genes related to squalene synthesis (ERG8, IDI1, MVD1), and genes related to cell growth (ERG12, GAL1, OCA1) were selected and overexpressed in strains. Using YOS5 as the starting strain, the effect of overexpression of each gene on squalene yield was verified by shake-flask fermentation experiments, and key genes with yield-increasing potential were identified. The experimental results are as follows.

[0126] (1) Verification of overexpression of yellow water adaptation-related genes.

[0127] Using the YOS5 strain as a control (squalene yield 430.7 mg / L), overexpression of three genes related to yellow water adaptability—ADH1, FBP1, and FDH1—was validated. The results are as follows: Figure 11 As shown in the figure. Experimental results indicated that the effects of different gene overexpressions on squalene production varied significantly (P<0.05): overexpression of ADH1 resulted in *Saccharomyces cerevisiae* YOS11, with a squalene production increase to 471.3 mg / L, a 9.43% increase compared to the control strain; overexpression of FBP1 resulted in *Saccharomyces cerevisiae* YOS12, with a squalene production increase to 464.2 mg / L, a 7.78% increase compared to the control strain; while overexpression of FDH1 resulted in *Saccharomyces cerevisiae* YOS13, with both squalene production and cell synthesis efficiency decreasing. In conclusion, overexpression of ADH1 and FBP1 can significantly increase squalene production and has the potential for further combination optimization.

[0128] (2) Verification of overexpression of squalene synthesis-related genes.

[0129] Using the YOS5 strain as a control (squalene yield 430.7 mg / L), overexpression of three squalene synthesis-related genes, ERG8, IDI1, and MVD1, was validated. The results are as follows: Figure 12 As shown in the figure. Experimental results showed that overexpression of ERG8 and IDI1 significantly increased squalene production: *Saccharomyces cerevisiae* strain YOS14, obtained after overexpression of ERG8, achieved an average squalene yield of 508.69 mg / L, an increase of 18.12% compared to the control strain, demonstrating the most significant improvement; *Saccharomyces cerevisiae* strain YOS15, obtained after overexpression of IDI1, achieved a squalene yield of 464.2 mg / L, an increase of 7.78% compared to the control strain; while *Saccharomyces cerevisiae* strain YOS16, obtained after overexpression of MVD1, although showing improved efficiency in squalene synthesis, ultimately experienced a decrease in squalene production. Therefore, ERG8 and IDI1 are key genes promoting squalene synthesis and can serve as core targets for subsequent combined overexpression.

[0130] (3) Verification of overexpression of bacterial growth-related genes.

[0131] Using the YOS5 strain as a control (squalene yield 430.7 mg / L), overexpression of three cell growth-related genes, ERG12, GAL1, and OCA1, was validated. The results are as follows: Figure 13 As shown in the figure. Experimental results showed that the effects of overexpression of different cell growth-related genes on squalene production varied significantly (P<0.05): Overexpression of OCA1 resulted in *Saccharomyces cerevisiae* YOS19, with a squalene yield of 450.25 mg / L, a 4.54% increase compared to the control strain. This is presumably because OCA1 overexpression improved cell membrane protein assembly and mitochondrial function, promoting cell growth and indirectly increasing squalene production. However, overexpression of GAL1 and ERG12 in *Saccharomyces cerevisiae* YOS18 and YOS17 both showed a significant decrease in squalene production. It is speculated that GAL1 overexpression may lead to excessive carbon source shift to glucose metabolism, and ERG12 overexpression may cause metabolic imbalance in the MVA pathway, both of which are detrimental to squalene accumulation. Therefore, only OCA1 overexpression has a certain potential for increasing squalene production and can be used as an auxiliary optimization target.

[0132] The results of single-gene overexpression showed that ERG8, ADH1, FBP1, IDI1 and OCA1 could all promote squalene accumulation to varying degrees, with ERG8 showing the most significant effect, indicating that it had a stronger effect on increasing the flux of squalene synthesis.

[0133] (4) Verification of co-expression of the target gene.

[0134] Based on the validation of single-gene overexpression, this invention selected ADH1, OCA1, FBP1, IDI1, and ERG8, which showed good yield-increasing effects, for combined co-expression to evaluate the impact of multi-gene synergistic regulation on squalene synthesis and to screen for the optimal gene combination. The results are as follows: Figure 14The results of fermentation after ADH1 and OCA1 were simultaneously integrated into YOS5 to obtain strain YOS20 showed that the average squalene yield of strain YOS20 was 479.27 mg / L, which was not further improved compared with strains that overexpressed ADH1 and OCA1 single genes, indicating that this combination did not produce a significant synergistic yield-increasing effect. After ADH1 and FBP1 were simultaneously integrated into YOS5, the squalene yield of strain YOS21 was 444.15 mg / L, which was lower than that of the two single-gene overexpression strains, indicating that this combination did not form a positive synergistic effect and may have disturbed cellular carbon metabolism allocation and energy metabolism balance, which is not conducive to squalene accumulation. In contrast, after ADH1 and IDI1 were simultaneously integrated into YOS5, the squalene yield of strain YOS22 was further improved to 502.6 mg / L, indicating that ADH1 and IDI1 had a good synergistic promoting effect. Among them, IDI1 helps maintain the balance between IPP and DMAPP and improve the utilization efficiency of downstream precursors; ADH1 can improve the intracellular reducing power state and optimize carbon flux distribution, thereby jointly promoting squalene synthesis.

[0135] Building upon this, ERG8 was further integrated into YOS22 to obtain the YOS23 tri-gene co-expression strain. The results are as follows... Figure 15 The results showed that the strain achieved an average squalene yield of 559.07 mg / L, which was further improved compared to the dual-gene combination and significantly higher than that of each single-gene overexpression strain (P<0.05), indicating that the introduction of ERG8 can further enhance the squalene biosynthetic flux. Comprehensive analysis suggests that IDI1, ERG8, and ADH1 act on key processes such as precursor balance, intermediate transformation, and reducing power and carbon flux partitioning, respectively. Their combined expression can synergistically optimize metabolic flux, thereby achieving a higher level of squalene accumulation.

[0136] 1.3.9 Results of single-factor fermentation experiments.

[0137] Using the optimal strain YOS23 with co-expression of three genes as the research object, the effects of fermentation time, yellow water concentration, galactose concentration, pH and rotation speed on squalene yield were investigated through single-factor experiments to determine the optimal level of each factor, laying the foundation for subsequent orthogonal experiments.

[0138] (1) Effect of fermentation time on squalene production by YOS23 engineered bacteria.

[0139] The growth curve and squalene production of the YOS23 engineered bacteria in yellow water liquid culture medium were dynamically monitored, such as... Figure 16 The growth curve of the strain exhibits typical microbial growth patterns: 0–12 h is the lag phase, and the strain's OD... 600 Growth is slow, and the bacteria are in the adaptation phase; the logarithmic growth phase occurs from 12 to 24 hours, and the OD of the strain... 600The growth rate increases rapidly, with a large proliferation of cells. The period from 24 to 96 hours is the stationary phase, during which the strain's growth stabilizes, metabolic activity is vigorous, and squalene continues to accumulate. After 96 hours, the decline phase begins, with a gradual decrease in cell count, weakened metabolic capacity, and a decline in squalene production. Based on the squalene production variation pattern, the squalene production reaches its peak at 96 hours of fermentation. Therefore, the optimal fermentation time for YOS23 engineered bacteria in yellow water is determined to be 96 hours.

[0140] (2) Effect of yellow water concentration on the production of squalene by YOS23 engineered bacteria.

[0141] Yellow water, used as a fermentation medium, not only contains substances necessary for microbial growth but also contains organic acids, tannins, and phenolic substances that can inhibit biological growth. Therefore, excessively high or low concentrations of yellow water may affect strain growth and squalene synthesis. Results are as follows... Figure 17 The results showed that the concentration of the yellow liquid had a significant impact on squalene yield: the lowest squalene yield was observed at a yellow liquid concentration of 10%, at only 390.1 mg / L; the highest yield was observed at a yellow liquid concentration of 80%, at 713.5 mg / L; further increasing the yellow liquid concentration to 100% did not result in a significant increase in squalene yield, but rather a slight decrease, possibly because excessively high yellow liquid concentrations may introduce too many inhibitory substances (such as formic acid and higher alcohols). Therefore, the optimal yellow liquid concentration for YOS23 engineered bacteria fermentation was determined to be 80%.

[0142] (3) Effect of galactose concentration on squalene production by YOS23 engineered bacteria.

[0143] The results are as follows Figure 18 The results showed that galactose concentration had a certain impact on squalene yield (P<0.05): the lowest squalene yield (500.1 mg / L) was observed at a galactose concentration of 2 g / L; as the galactose concentration increased, the squalene yield gradually increased, reaching a maximum of 633.2 mg / L at a galactose concentration of 10 g / L; the squalene yields at 6 g / L and 8 g / L were 578.1 mg / L and 618.0 mg / L, respectively, with small differences compared to the yield at 10 g / L (differences of 19.1 mg / L and 5.2 mg / L, respectively). From the perspective of maximizing yield, 10 g / L is the optimal concentration, but from the perspective of cost-effectiveness and resource conservation, 80 g / L is a better choice.

[0144] (4) Effect of pH on squalene production by YOS23 engineered bacteria.

[0145] The pH value of the fermentation medium directly affects the activity of yeast, cell membrane stability, and the absorption and utilization of nutrients, as shown in the following results. Figure 19As shown, pH has a significant impact on squalene yield (P<0.05): the highest squalene yield (588.1 mg / L) was observed at pH=6, when the bacterial enzyme activity was strongest, cell membrane stability was optimal, and metabolic flux was highly efficient, which was conducive to squalene synthesis. The lowest squalene yield (381.9 mg / L) was observed at pH=8, and the yield was also low at pH=3.8. Therefore, the optimal pH for fermentation of the YOS23 engineered bacteria was determined to be 6.

[0146] (5) Effect of rotation speed on squalene production by YOS23 engineered bacteria.

[0147] The shaking speed affects the dissolved oxygen level and mass transfer efficiency of the fermentation system, thereby regulating cell growth and squalene synthesis. For example... Figure 20 As shown, squalene production exhibited a significant trend of first increasing and then decreasing with increasing rotational speed (P<0.05). The highest squalene production (525 mg / L) was achieved at 220 r / min, indicating that an appropriate rotational speed can improve dissolved oxygen conditions and promote product accumulation. However, when the rotational speed continued to increase to 250 r / min and 280 r / min, squalene production decreased, possibly due to excessive shearing affecting normal cell metabolism and metabolic flux distribution. Therefore, the optimal fermentation speed for YOS23 engineered bacteria was determined to be 220 r / min.

[0148] 1.3.10 Results of orthogonal experiment on the effect of yellow water concentration on YOS23 engineered bacteria.

[0149] Based on the results of single-factor experiments, orthogonal experiments were conducted to optimize the squalene yield by selecting yellow water concentration, pH value, and rotation speed. Intuitive analysis of the experimental results showed that, within the experimental design range, the optimal experimental scheme was: yellow water concentration of 80%, pH value of 6, and rotation speed of 220 r / min. Analysis of variance showed that yellow water concentration had a significant impact on yield, while pH had a relatively smaller effect on squalene yield.

[0150] Single-factor and orthogonal experiments determined the optimal fermentation process for strain YOS23. The optimal yield was 740.4 mg / L with a galactose concentration of 80 g / L, a yellow water concentration of 80%, a pH of 6, and a rotation speed of 220 r / min. This achieved efficient utilization of the yellow water and increased squalene production.

[0151] 1.3.11 COD removal rate before and after fermentation.

[0152] To investigate the organic matter removal effect of the yellow water fermentation system under the optimal combination conditions of the orthogonal experiment, the COD of the culture solution before and after fermentation was measured, and the COD removal rate (%) was calculated as follows: (COD before fermentation − COD after fermentation) / COD before fermentation × 100%. The results are shown in Table 9, and the average removal rate is 58.33%. Combining the COD change results before and after fermentation, it can be seen that the fermentation process of Saccharomyces cerevisiae has a certain reduction effect on the organic load in the yellow water, indicating that while this system realizes the biosynthesis of squalene, it also has a certain potential for reducing waste liquid emissions.

[0153] Table 9 Changes in COD and removal rate before and after fermentation 2. Study on the alleviating effect of high-squalene-producing Saccharomyces cerevisiae on DSS-induced colitis in mice.

[0154] Taking the DSS-induced mouse enteritis model as the research object, it was intervened with the disrupted product of high-squalene-producing Saccharomyces cerevisiae YOS5 from the common culture medium, or the disrupted product of high-squalene-producing Saccharomyces cerevisiae YOS23 from the yellow water culture system. Combining indicators such as inflammatory factors, oxidative stress levels, and pathological changes in colon and liver tissues, its alleviating effect on mouse enteritis and probiotic potential were systematically evaluated.

[0155] 2.1 Experimental materials.

[0156] 2.1.1 Test strains.

[0157] Saccharomyces cerevisiae YOS1, Saccharomyces cerevisiae YOS5, Saccharomyces cerevisiae YOS23.

[0158] 2.1.2 Test animals.

[0159] Healthy male C57BL / 6N mice, 6 weeks old, were provided by Hubei Beite Biotechnology Co., Ltd., and the experimental animal production license number was SCXK[E]2020-0018. The mice were housed in the SPF environment of the Experimental Animal Center of Hubei Normal University, and the feeding conditions were temperature 22 ± 2 °C, relative humidity 50%, 12 h light-dark cycle, and free access to food and water. Before the formal experiment, all mice were adaptively fed for 3 d.

[0160] 2.1.3 Reagents and solutions.

[0161] ELISA kits (IL-1β), ELISA kits (IL-6), ELISA kits (TNF-α), micromalondialdehyde (MDA) kits, superoxide dismutase (SOD) assay kits, glutathione peroxidase (GSH-Px) assay kits, and catalase (CAT) assay kits were purchased from Wuhan Sewell Biotechnology Co., Ltd. DSS (dextran sulfate sodium) and SASP were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Methanol, acetonitrile, and formic acid were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0162] 2.2 Experimental methods.

[0163] 2.2.1 Preparation of Saccharomyces cerevisiae by gavage fermentation in mice.

[0164] Saccharomyces cerevisiae YOS5 and YOS1 were prepared by gavage and fermentation. The Saccharomyces cerevisiae were inoculated into YPD seed culture medium and cultured for 1 day. Then, they were transferred to YPG medium at an inoculation rate of 16% and cultured with shaking at 30℃ and 220 r / min for 96 h. After fermentation, the fermentation broth was collected, centrifuged, and the supernatant was discarded. The cells were washed with bacterial water and resuspended. The resuspended liquid was then crushed using a high-pressure homogenizer.

[0165] The preparation method of the yellow water fermented brewing yeast sample by gavage is basically the same as that of ordinary brewing yeast. The sample is prepared for gavage, stored at 4℃ for later use, and brought to room temperature and thoroughly mixed before use.

[0166] 2.2.2 Animal experiment design.

[0167] The isolation and screening of Saccharomyces cerevisiae and its efficacy in alleviating colitis in mice were conducted at the School of Life Sciences, Hubei Normal University. The experimental procedure has been ethically reviewed (ethics number: HBNUSTER-202501). All C57BL / 6 mice were housed in an SPF (specific pathogen-free) environment with a temperature of 22-24℃ and a relative humidity of 50-60%, with a normal diet and unlimited access to drinking water.

[0168] (1) YOS5, a brewer's yeast that overexpresses ERG9 and weakens ERG1.

[0169] Control group (NC): During the experimental period, the control group had free access to normal drinking water and was given physiological saline by gavage daily.

[0170] DSS model group (DSS): Mice were allowed free access to 3% DSS solution starting on day 4, and were given an equal volume of physiological saline by gavage daily. On day 8, they were allowed free access to normal drinking water.

[0171] YOS5 Saccharomyces cerevisiae intervention group (REC): Based on the 3% DSS free drinking water model, YOS5 Saccharomyces cerevisiae was administered by gavage daily at a dose of 0.4 mL.

[0172] YOS1 intervention group (FPP): Based on the 3% DSS free drinking water model, YOS1 yeast was administered by gavage daily at a dose of 0.4 mL.

[0173] (2) High-yield squalene-producing brewer's yeast YOS23 for yellow water utilization.

[0174] Blank control group (KB): During the experimental period, participants had free access to normal drinking water and were given physiological saline by gavage daily.

[0175] DSS model group (DSS): Mice were allowed free access to 3% DSS solution starting on day 4, and were given an equal volume of physiological saline by gavage daily. On day 9, they were allowed free access to normal drinking water.

[0176] YOS23 (HG) group: Based on the modeling of free drinking water with 3% DSS, YOS23 brewer's yeast fermented with yellow water was administered by gavage daily at a dose of 0.4 mL.

[0177] YOS1 (HF) group: Based on the modeling of free drinking water in 3% DSS, common brewer's yeast fermented with yellow water was administered by gavage daily at a dose of 0.4 mL.

[0178] Positive drug group (YY): Based on the 3% DSS free drinking water model, SASP was administered by gavage daily at a dose of 0.4 mL.

[0179] 2.2.3 Measurement of physiological indicators in mice.

[0180] During the experiment, the body weight of mice in each group was recorded daily, and the mice were scored for diarrhea, fecal morphology, and occult blood. The Disease Activity Index (DAI) was calculated based on these scores. Table 10 shows the corresponding scoring criteria for DAI.

[0181] Table 10 DAI Scoring Table 2.2.4 Mouse dissection and tissue collection.

[0182] After the experiment, mice were euthanized by enucleation of the eyeballs, and the samples were dissected and preserved. ① Blood: Blood was collected from the eyeballs, left for 1 hour, centrifuged at 3000 rpm for 15 minutes at 4℃, flash-frozen in liquid nitrogen, and stored at -80℃ for later use. ② Colon: The colon from the end of the cecum to the end of the anus was taken, photographed and its length recorded. The colon was simply divided into three parts (the upper part of the colon was fixed with 4% paraformaldehyde, one part of the remaining two parts was preserved in Trizol, and the other part was flash-frozen in liquid nitrogen and stored at -80℃). ③ Spleen: The spleen was weighed and the spleen index was calculated as (spleen weight / body weight × 100%). ④ Feces: The cecal tissue was longitudinally dissected, and the contents of the mouse cecum were squeezed into 1.5 mL sterile centrifuge tubes using sterile forceps. The tubes were numbered, flash-frozen in liquid nitrogen, stored at -80℃, and immediately sent for analysis.

[0183] 2.2.5 PAS staining of mouse colon.

[0184] A 0.5 cm section of colonic tissue was fixed in 4% paraformaldehyde. The tissue was then sent to Wuhan Saiweier Biotechnology Co., Ltd. for PAS staining of mouse colon.

[0185] 2.2.6 Determination of inflammatory cytokines and oxidative stress levels in mouse colon.

[0186] Whole blood samples should be left at room temperature for 2 hours, then centrifuged at 3000 r / min for 15 min at 2-8℃. The supernatant should be aliquoted and stored at -20℃ or -80℃, avoiding repeated freeze-thaw cycles. Thawed samples should be centrifuged again before testing. Serum TNF-α, IL-1β, and CRP levels were determined using ELISA, while T-SOD, MDA, and GSH were determined biochemically. The testing was performed by Sewell Biotechnology Co., Ltd.

[0187] 2.2.7 Determination of short-chain fatty acids in mouse feces.

[0188] (1) Mouse fecal preparation: Accurately weigh 0.4 g of mouse fecal sample, add 2 mL of sterile water, vortex mix for 30 s, and let stand in an ice bath for 5 min; then centrifuge at 4℃ and 12000 r / min for 10 min, transfer 1 mL of supernatant to a clean 1.5 mL centrifuge tube, add 10 μL of formic acid for acidification, and store at −20℃ for testing. Before instrumental analysis, take 600 μL of supernatant and filter it through a 0.22 μm organic filter membrane.

[0189] (2) Gas chromatograph conditions: The gas chromatograph used was equipped with an autosampler and a flame ionization detector. The chromatographic column was a highly polar DB-WAX 250 fused silica capillary column (30 m x 0.32 mm, 0.25 μm). The program was set as follows: initial temperature 70℃ held for 1 min, temperature increased to 110℃ at 10℃ / min, held for 1 min, temperature increased to 120℃ at 3℃ / min, held for 1 min, and finally temperature increased to 135℃ at 3℃ / min; injection port temperature 250℃, detector temperature 290℃, split ratio 10:1, injection volume 5 μL, and total duration 18.66 min.

[0190] 2.2.8 Non-targeted metabolomics assay in mouse feces.

[0191] On the day of dissection, the contents of the mouse colon were ground in liquid nitrogen, transported on dry ice, and delivered to Wuhan Sangon Biotech Co., Ltd. for non-targeted metabolomics sequencing.

[0192] 2.2.9 Determination of intestinal flora in mouse feces at 16s.

[0193] On the day of dissection, the contents of the mouse colon were ground in liquid nitrogen, transported on dry ice, and delivered to Shanghai Baipu Biotechnology Co., Ltd. for microbial diversity analysis.

[0194] 2.2.10 Data processing.

[0195] Raw data underwent peak alignment, retention time correction, and peak area extraction using MSDIAL software. Metabolite structure identification employed precise mass number matching (mass tolerance < 10 ppm) and secondary spectrum matching (mass tolerance < 0.01 Da), searching public databases such as HMDB, MassBank, and GNPS, as well as the self-built BP-DB metabolite library. For the extracted data, ion peaks with more than 50% missing values ​​within a group were removed and excluded from subsequent statistical analysis. The total peak area of ​​both positive and negative ion data was normalized separately. Positive and negative ion peaks were integrated, and pattern recognition was performed using Python software. Data was preprocessed using unit variance scaling (UV) before subsequent data analysis.

[0196] 2.3 Results and Analysis.

[0197] 2.3.1 Engineered strain YOS5 that overexpresses ERG9 and weakens ERG1.

[0198] (1) Changes in body weight.

[0199] The Disease Activity Index (DAI) is a composite score based on three factors: weight, rectal bleeding, and stool characteristics. For example... Figure 21As shown in Figures A and B, the body weight of mice in the NC group remained relatively stable, and their DAI scores remained consistently low, indicating a generally good condition. In the DSS group, body weight gradually decreased after modeling, while the DAI score continued to rise, indicating that the mice had developed obvious colitis symptoms, demonstrating the successful establishment of the model. Compared to the DSS group, the RCE group showed a smaller decrease in body weight and maintained a lower DAI score, demonstrating a more significant allergic effect. The FPP group also improved the DSS-induced body weight loss and DAI increase to some extent, but its overall effect was weaker than that of the RCE group. Overall, both types of Saccharomyces cerevisiae interventions could alleviate colitis symptoms in mice, with the high-squalene-producing Saccharomyces cerevisiae group showing a more significant improvement.

[0200] (2) Effects on the spleen of UC mice.

[0201] As an important immune organ, changes in the spleen's weight can reflect the body's systemic inflammatory state. For example... Figure 22 As shown in A and B, the spleen index of mice significantly increased after DSS modeling, indicating that the model was successfully constructed. Saccharin-producing Saccharin-producing Saccharin-producing group reduced the spleen index to varying degrees, with the improvement effect being significantly better than that of ordinary Saccharin-producing Saccharin-producing group fermented on ordinary culture medium, indicating that it has a better effect in alleviating inflammation and improving the body's immune status.

[0202] (3) Effects on the colon of UC mice.

[0203] In colitis models, colonic shortening is a typical characteristic of the degree of intestinal inflammation, and the degree of shortening is positively correlated with the severity of inflammation. Therefore, colonic length is the most intuitive and reliable indicator for assessing the condition of colitis. Figure 23 As shown, the normal control group mice had normal colon length, a smooth and uniform intestinal wall surface, normal color, and formed feces without edema, congestion, or other inflammatory signs. In contrast, the DSS-induced model group mice showed significant colon shortening, a dull intestinal wall surface, unformed feces, and obvious congestion, swelling, and other inflammatory damage. Compared to the model group, the high-squalene-producing Saccharomyces cerevisiae group showed varying degrees of improvement in colon length shortening, swelling, and congestion, with feces returning to a formed state. While the ordinary Saccharomyces cerevisiae group showed some improvement compared to the DSS group, the degree of recovery was relatively weak. This indicates that gavage administration of high-squalene-producing Saccharomyces cerevisiae can effectively alleviate DSS-induced colon shortening in mice and reduce intestinal inflammatory damage.

[0204] like Figure 24As shown, the colonic tissue of mice in the modeling group was severely damaged, with large ulcers, almost complete disappearance of crypts, and diffuse distribution of inflammatory cells. In the high-squalene-producing Saccharomyces cerevisiae intervention group, the colonic tissue of mice showed clear structure in each layer, relatively intact crypts, and no obvious inflammatory cell infiltration. Similarly, it was found that gavage with ordinary Saccharomyces cerevisiae could also slightly alleviate colonic tissue damage in mice. Pathological scoring results indicated that high-squalene-producing Saccharomyces cerevisiae could improve the pathological damage in mice with DSS-induced colitis.

[0205] (4) Biochemical experimental results.

[0206] like Figure 25 As shown in Figures A to C, compared with the normal control group, the serum MDA level in the model group mice increased slightly by 0.70%, T-SOD activity decreased by 46.96%, and GSH content decreased by 11.26%, indicating that the antioxidant defense capacity of mice was weakened and the level of oxidative stress increased after DSS treatment, suggesting that the model group had significant oxidative damage, indicating that the colitis model was successfully established. After intervention with high-squalene-producing Saccharomyces cerevisiae, the serum MDA level in mice decreased by 9.75% compared with the model group, T-SOD activity increased by 51.43%, and GSH content increased by 17.90%, indicating that this treatment can effectively improve the body's oxidative stress state, enhance free radical scavenging capacity, and reduce lipid peroxidation damage. After intervention with high-FPP Saccharomyces cerevisiae, the serum MDA level in mice decreased by 25.04% and GSH increased by 14.99% compared with the model group, but T-SOD decreased slightly by 2.49% compared with the model group, indicating that this treatment was more effective in reducing lipid peroxidation levels, but its effect on improving antioxidant enzyme activity was not significant.

[0207] (5) ELISA experimental results.

[0208] like Figure 26 As shown in Figures A to C, compared with the normal control group, the serum levels of IL-1β, TNF-α, and CRP in the DSS group mice were significantly increased, increasing by 20.82%, 214.06%, and 33.84%, respectively, indicating that a significant inflammatory response had been established in the model group, demonstrating the successful construction of the colitis model. After intervention with high-squalene-producing Saccharomyces cerevisiae, all of the above inflammatory markers decreased compared with the model group, with IL-1β, TNF-α, and CRP decreasing by 6.57%, 59.09%, and 20.58%, respectively, suggesting that this treatment can inhibit the release of inflammatory factors to a certain extent and reduce inflammatory damage in the body. In contrast, after intervention with high-FPP-producing Saccharomyces cerevisiae, serum IL-1β, TNF-α, and CRP increased by 34.77%, 78.18%, and 8.39%, respectively, compared with the model group, without showing an anti-inflammatory effect. This indicates that under the conditions of this experiment, non-squalene-producing FPP engineered bacteria are unlikely to improve the DSS-induced inflammatory state and may even further aggravate the inflammatory response in the body.

[0209] In summary, the high-squalene-producing Saccharin-producing Saccharin-producing yeast has a good anti-inflammatory effect on DSS-induced enteritis mice, indicating that squalene may play an important role in it, and also providing a basis for subsequent development of functional strains and nutritional intervention research for enteritis.

[0210] (6) Effects on the content of short-chain fatty acids in UC mice.

[0211] like Figure 27 As shown in Figures A to C, the fecal levels of butyric acid, propionic acid, and acetic acid in the NC group mice were all at high levels. After DSS modeling, the levels of all three short-chain fatty acids decreased significantly, indicating that the intestinal flora's ability to ferment and produce acid may be weakened under colitis conditions, and intestinal metabolic function may be significantly impaired. Compared with the DSS group, the levels of short-chain fatty acids in the REC and FPP groups showed varying degrees of recovery, indicating that yeast intervention can improve intestinal metabolic disorders to some extent. Among them, the REC group was generally better than the FPP group in terms of the recovery of butyric acid, propionic acid, and acetic acid. These results indicate that high-squalene-producing Saccharin-producing Saccharin-producing yeast is more effective than ordinary yeast in promoting the production of beneficial metabolites and improving intestinal microecological imbalance, thus playing a better role in alleviating DSS-induced intestinal damage. This suggests that SCFAs played a positive regulatory role as a positive regulator during the improvement of colitis in mice by high-squalene-producing Saccharin-producing Saccharin-producing yeast.

[0212] (7) Effects on the non-targeted metabolome of UC mice.

[0213] The differentially metabolites mainly involved lipids and lipid molecules, organic acids and their derivatives, benzene ring compounds, and nucleosides. These metabolites are closely related to inflammatory responses, energy metabolism, and gut microbiota function. The REC group showed better overall adjustment in these key metabolic categories than the FPP group, suggesting that squalene may play a positive role in improving intestinal inflammation and promoting metabolic homeostasis. The differentially metabolites were mainly concentrated in pathways related to amino acid metabolism, energy metabolism, carbohydrate metabolism, and cofactor metabolism. Overall, the differentially metabolites primarily reflect the remodeling of the metabolic network under inflammatory stress, indicating that the Saccharomyces cerevisiae treatment group may exert its effects by regulating amino acid metabolism, energy supply, and gut-related metabolic processes.

[0214] In summary, the anti-inflammatory and intestinal protective effects of high-squalene-producing Saccharomyces cerevisiae YOS5 derived from ordinary culture medium have been preliminarily elucidated, and the relevant mechanism of action has been clarified. This lays a solid foundation for subsequent functional evaluation and safety studies of high-squalene-producing Saccharomyces cerevisiae fermented in yellow water culture medium.

[0215] 2.3.2 Study on the effect of fermentation of Saccharomyces cerevisiae YOS23 on DSS-induced colitis in mice.

[0216] (1) Changes in body weight.

[0217] For the results of weight change, as follows Figure 28 Figures A and B show that the body weight of mice in each group was basically stable before modeling, with no significant differences between groups. After DSS treatment, the body weight of mice in the model group gradually decreased, suggesting that modeling led to damage to the body's condition. Compared with the DSS group, the trend of body weight loss in each intervention group was alleviated. Among them, the HG group had smaller overall fluctuations and relatively better body weight maintenance, followed by the HF group, indicating that high-squalene-producing Saccharomyces cerevisiae has a certain ameliorative effect on DSS-induced body weight loss in mice.

[0218] (2) Effects on the spleen of UC mice.

[0219] like Figure 29 As shown in Figures A and B, the spleen index of mice significantly increased after DSS treatment, indicating the successful establishment of the colitis model. After different treatments, the spleen index of mice in each intervention group decreased compared to the DSS group. Specifically, the spleen index of the HG group was 0.22560, a decrease of 36.82% compared to the DSS group, and approached the level of the KB group; the spleen indices of the HF and YY groups were 0.28277 and 0.28540, respectively, a decrease of 20.81% and 20.08% compared to the DSS group. The experimental results indicate that different treatments can alleviate the DSS-induced increase in spleen index to some extent, with the HG group showing the most significant improvement. This suggests that high-squalene-producing *Saccharin-producing* *Saccharin-producing* yeast may have a better effect in reducing systemic inflammatory responses and improving abnormalities in immune organs.

[0220] (3) Effects on the colon of UC mice.

[0221] Figure 30 It was found that, based on the gross morphology of the colons in each group of mice, the colon in the KB group appeared normal. The colon in the DSS group was significantly shortened, with thickened intestinal segments, and noticeable congestion and fecal retention were observed, indicating severe inflammatory damage to the colon after DSS treatment, suggesting a relatively ideal model establishment. Compared to the DSS group, the colon length in the HG, HF, and YY groups all showed varying degrees of recovery, and the tissue damage was reduced. This indicates that the high-squalene-producing Saccharin-producing Saccharin-producing yeast fermented in yellow water can alleviate DSS-induced colonic damage to a certain extent and has a good ameliorative effect on colonic shortening.

[0222] (4) Effects on the content of short-chain fatty acids in the feces of UC mice.

[0223] This invention further examined the changes in short-chain fatty acid content in the feces of mice in different treatment groups to evaluate the effect of Saccharomyces cerevisiae intervention on intestinal metabolic function. The results are as follows: Figure 31As shown in Figures A to C, the levels of various short-chain fatty acids in the feces of mice in the DSS model group were significantly reduced compared to the blank control group. Compared to the DSS group, the levels of major short-chain fatty acids such as acetic acid, propionic acid, and butyric acid in the feces of mice in the HG group were increased, and their trends were similar to those of the positive drug group. Although the HF group also showed a certain degree of increase, the overall improvement effect was relatively weak.

[0224] (5) Effects on gut microbiota in UC mice.

[0225] ① Venn diagram.

[0226] like Figure 32 As shown, the five groups shared a total of 216 OTUs, accounting for 2.46%, indicating the existence of a relatively stable core microbiota among the groups. The OTUs specific to the KB, DSS, HG, HF, and YY groups accounted for 22.49%, 18.82%, 15.84%, 15.46%, and 13.40%, respectively. Among the intervention groups, the HG group had the highest proportion of specific OTUs, exceeding the HF and YY groups by 0.38 and 2.44 percentage points, respectively. This indicates that the HG group formed more characteristic microbiota during the gut microbiota remodeling process, and the overall adjustment magnitude was better than that of the HF and YY groups. Combined with the shared OTU results, the HG group demonstrated a stronger microbiota reconstruction capacity while retaining the core microbiota, suggesting a relatively better effect on improving the gut microbiota.

[0227] ② Level of gut microbiota in mice.

[0228] At the level of the door, such as Figure 33 As shown, the gut microbiota of each group mainly consisted of Bacteroidetes, Firmicutes, and Verrucous microbes. Compared with the KB group, the DSS group showed a decrease in the relative abundance of Bacteroidetes, an increase in Firmicutes, and a decrease in Verrucous microbes, indicating that the gut microbiota structure of mice was disordered after DSS induction. After intervention, both the HG and HF groups showed some improvement in the microbiota imbalance, with the HG group showing a more significant increase in Verrucous microbes and an overall regulatory effect superior to the HF group; the microbiota structure of the YY group was closer to that of the normal group.

[0229] ③ Level of gut microbiota in mice.

[0230] From the perspective of subordinate level, such as Figure 34 As shown in Figures A to G, DSS treatment significantly altered the gut microbiota structure in mice, resulting in an increase in Akkermania spp. ( Akkermansia The proportion of certain dominant bacterial genera decreased significantly, while the proportions of some other dominant bacterial genera fluctuated, indicating a disruption of the gut microbiota balance. After intervention, the composition of the gut microbiota in all treatment groups showed varying degrees of recovery, with the HG group showing the best results. Akkermansia The improvement was most significant, demonstrating a strong ability to repair the intestinal mucosa-associated flora; at the same time Allobaculum, Odoribacter, ButyricicoccusOther bacterial genera closely related to gut health also showed an upward trend. This indicates that *Saccharin-producing* *Saccharin-producing* yeast from Huangshui can more effectively regulate DSS-induced gut microbiota imbalance and promote the restoration of the microbiota structure to a relatively healthy state.

[0231] (6) Effects on the non-targeted metabolome of UC mice.

[0232] In addition to its inherent regulatory functions, the high-squalene-producing *Saccharinus sacchariformis* may exhibit more significant advantages in lipid metabolism regulation, antioxidant activity, and anti-inflammatory effects due to squalene enrichment. Differential metabolites mainly involve pathways related to environmental information processing, metabolism, and bodily systems, with the sphingolipid signaling pathway, phospholipase D signaling pathway, ABC transporter, tryptophan metabolism, pyruvate metabolism, citric acid cycle, and multiple amino acid metabolic pathways being particularly prominent. In conclusion, the high-squalene-producing *Saccharinus sacchariformis* YOS23, fermented in yellow water, demonstrates good intervention effects and reliable safety in mouse colitis, and is superior to *Saccharinus sacchariformis* YOS1, providing experimental evidence for its use as a green feed additive and the high-value utilization of yellow water resources.

[0233] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A recombinant brewing yeast that produces high levels of squalene, characterized in that, The recombinant Saccharomyces cerevisiae is based on Saccharomyces cerevisiae YOS1, with overexpression of the squalene synthase ERG9 gene and weak expression of the squalene epoxidase ERG1 gene in Saccharomyces cerevisiae YOS1. The integration site of the squalene synthase ERG9 gene is the 1021b site of the genome of the Saccharomyces cerevisiae YOS1. The specific method for weak expression of the squalene epoxidase ERG1 gene is as follows: the promoter of the squalene epoxidase ERG1 gene of the Saccharomyces cerevisiae YOS1 is replaced with a weak promoter. The weak promoter is the REV1 gene promoter; The sequence of the squalene synthase ERG9 gene is shown in SEQ ID NO: 51; The sequence of the squalene epoxidase ERG1 gene is shown in SEQ ID NO: 52; The *Saccharomyces cerevisiae* YOS1 strain is an engineered *Saccharomyces cerevisiae* strain with the genotype erg9::KanMX / CTR3p-ERG9;leu2-3_112::His3MX6 / GAL1p-ERG19 / GAL10p-ERG8;ura3-52::GAL1p-EfMvaS(A110G)-CYC1t / GAL10p-EfMvaE-ADH1t;his3Δ1::hphMX4 / GAL1p-ERG12 / GAL10p-IDI1;1014a::GAL1p-Erg20.

2. The recombinant brewing yeast with high squalene production according to claim 1, characterized in that, The recombinant Saccharomyces cerevisiae also overexpresses any one of the following genes: mevalonate phosphate kinase ERG8 gene, alcohol dehydrogenase ADH1 gene, fructose-1,6-bisphosphatase FBP1 gene, isoprenyl pyrophosphate isomerase IDI1 gene, and endoplasmic reticulum membrane protein complex subunit OCA1 gene. The sequence of the mevalonate kinase ERG8 gene is shown in SEQ ID NO: 53; The sequence of the alcohol dehydrogenase ADH1 gene is shown in SEQ ID NO: 54; The sequence of the fructose-1,6-bisphosphatase FBP1 gene is shown in SEQ ID NO: 55; The sequence of the isoprenyl pyrophosphate isomerase IDI1 gene is shown in SEQ ID NO: 56; The sequence of the OCA1 gene, a subunit of the endoplasmic reticulum membrane protein complex, is shown in SEQ ID NO: 57; The site at which the mevalonate kinase ERG8 gene integrates into the genome of the Saccharomyces cerevisiae YOS1 is int18; The site at which the alcohol dehydrogenase ADH1 gene integrates into the genome of the Saccharomyces cerevisiae YOS1 is int6; The site at which the fructose-1,6-bisphosphatase FBP1 gene integrates into the genome of the Saccharomyces cerevisiae YOS1 is int6; The site at which the isoprenyl pyrophosphate isomerase IDI1 gene is integrated into the genome of the Saccharomyces cerevisiae YOS1 is int4; The site at which the endoplasmic reticulum membrane protein complex subunit OCA1 gene integrates into the genome of the Saccharomyces cerevisiae YOS1 is int6.

3. The recombinant brewing yeast with high squalene production according to claim 1, characterized in that, The recombinant Saccharomyces cerevisiae also overexpresses the alcohol dehydrogenase ADH1 gene and the isoprenyl pyrophosphate isomerase IDI1 gene. Alternatively, the recombinant Saccharomyces cerevisiae may also overexpress the alcohol dehydrogenase ADH1 gene, the isoprenyl pyrophosphate isomerase IDI1 gene, and the mevalonate phosphate kinase ERG8 gene. The sequence of the alcohol dehydrogenase ADH1 gene is shown in SEQ ID NO: 54; the site at which the alcohol dehydrogenase ADH1 gene integrates into the genome of the Saccharomyces cerevisiae YOS1 is int6; The sequence of the isoprenyl pyrophosphate isomerase IDI1 gene is shown in SEQ ID NO: 56; the site at which the isoprenyl pyrophosphate isomerase IDI1 gene is integrated into the genome of the Saccharomyces cerevisiae YOS1 is int4; The sequence of the mevalonate kinase ERG8 gene is shown in SEQ ID NO: 53; the site at which the mevalonate kinase ERG8 gene is integrated into the genome of the Saccharomyces cerevisiae YOS1 is int18.

4. A method for producing squalene, characterized in that, The process includes the following steps: culturing the high-squalene-producing recombinant Saccharin yeast of any one of the following 1 to 3 in a culture medium, and collecting the squalene from the fermentation products; The culture medium includes YPG medium or pretreated yellow water culture medium, a byproduct of baijiu brewing. The pretreated culture medium for yellow water from baijiu brewing byproducts was prepared through the following steps: 2% (w / w) of chitosan with a pH of 4 was added to the yellow water from baijiu brewing byproducts, followed by 5% (w / w) of 12-mesh activated carbon for adsorption treatment. The mixture was then filtered to remove the activated carbon and large particulate impurities, yielding a filtrate. The pH of the filtrate was adjusted to 6, and the supernatant was collected by centrifugation and filtered through a 0.45 μm microporous membrane. Finally, 4% galactose was added to prepare the pretreated culture medium for yellow water from baijiu brewing byproducts.

5. A method for the resource utilization of yellow water, a byproduct of Baijiu (Chinese liquor) brewing, characterized in that, The method includes the following steps: pre-treating the yellow water, a by-product of baijiu brewing, and using it as a culture medium to cultivate the recombinant brewing yeast that produces high squalene as described in any one of claims 1 to 3, thereby producing squalene while reducing the chemical oxygen demand of the yellow water.

6. The use of the high-squalene-producing recombinant brewer's yeast as described in any one of claims 1 to 3, or the fermentation product prepared by the method for producing squalene as described in claim 4, in the preparation of a medicament for relieving colitis.

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