Saccharomyces cerevisiae strain with high glycerol yield, construction method and application thereof

Abstract

The present application belongs to the field of genetic engineering and microbial fermentation technology, and particularly relates to a construction method of a high-glycerol yeast strain and application thereof. The present application precisely predicts a key active site I184 of glycerol-3-phosphate dehydrogenase by a strategy combining rational design and scarless editing, and realizes site-specific scarless mutation of I184L in an industrial Saccharomyces cerevisiae by using CRISPR-Cas9 technology and homologous recombination, and successfully constructs an engineering strain G410-M184 strain. In grape juice fermentation, the glycerol yield of the mutant strain is significantly improved by about 45%-51% compared with the original strain. At the same time, the basic fermentation performance, growth rate and aroma spectrum of the strain are not adversely affected, and the excellent fermentation characteristics are completely retained. Therefore, the high-glycerol yeast strain constructed by the present application is particularly suitable for the brewing of grape wine and other fruit wine, can effectively enhance the roundness and fullness of the product, improve the quality, and has important industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and microbial fermentation technology, specifically relating to a method for constructing a high-glycerol-producing yeast strain and its application. Background Technology

[0002] The roundness and fullness of a wine's flavor are closely related to its glycerol content. Glycerol, a major byproduct of alcoholic fermentation, contributes to the sweetness and body of wine. Therefore, breeding high-glycerol-producing yeast strains has significant industrial application value for improving wine quality.

[0003] Currently, traditional methods for increasing glycerol production in yeast strains mainly include:

[0004] (1) Conventional mutagenesis breeding: High-yielding strains are obtained by random mutagenesis methods such as ultraviolet light and chemical mutagens, followed by high-throughput screening. This method is highly random, the mutation sites are unclear, and it is usually accompanied by multiple mutagenesis, which can easily lead to the decline of strain growth or other fermentation performance. The breeding cycle is long and the workload is large.

[0005] (2) Traditional genetic engineering modification: This involves overexpressing genes for key enzymes in glycerol synthesis (such as glycerol-3-phosphate dehydrogenase, GPD) to increase glycerol production. However, this leads to an increase in undesirable metabolites such as acetaldehyde, acetic acid, and diacetyl (acetoin), affecting the flavor of the wine. Moreover, this method usually relies on expression vectors that are randomly integrated into the genome or have multiple copies, which may disrupt genome stability and often requires the introduction of exogenous selection marker genes such as antibiotic resistance. This not only may pose biosafety risks but also limits its application in the food industry, especially in winemaking.

[0006] (3) Early site-directed mutagenesis technology: Although it can achieve mutation at specific sites, the operation process is cumbersome and the efficiency is low. It is also difficult to avoid the residue of foreign DNA sequences (such as selection markers), and it cannot achieve true "traceless" editing.

[0007] With the development of CRISPR-Cas9 gene editing technology, it has shown great potential in microbial breeding. However, how to rationally design and precisely construct a mutant strain that can significantly increase glycerol production without affecting other normal fermentation performance of yeast remains a pressing technical challenge in this field. Current technology lacks a method to efficiently construct high-glycerol-producing yeast strains through precise directed evolution of key enzymes without introducing exogenous genes. Summary of the Invention

[0008] In order to improve the roundness of wine, the present invention aims to provide a mutant strain of brewing yeast that produces high glycerol and has stable fermentation performance, its rationally designed construction method, and its application in winemaking.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a high-glycerol-producing mutant strain of Saccharomyces cerevisiae, classified as Saccharomyces cerevisiae G410-M184, whose glycerol-3-phosphate dehydrogenase gene (GPD1) contains the I184L point mutation.

[0011] Secondly, the present invention provides a method for constructing the high-glycerol-producing Saccharomyces cerevisiae mutant strain, comprising the following steps:

[0012] (1) By molecular docking and multiple sequence alignment, potential high-activity mutation sites of glycerol-3-phosphate dehydrogenase were rationally designed and screened. The potential high-activity sites are F73Y, I184L, T186P, R132K, and T305S.

[0013] (2) Using the haploid strain of Saccharomyces cerevisiae FY1679-01B, the candidate mutation sites obtained in step (1) were functionally verified in vivo through homologous recombination and reverse substitution technology, and the optimal mutation site I184L that can significantly increase glycerol production was screened out.

[0014] (3) In diploid industrial brewing yeast G410, the I184L mutation was precisely and seamlessly introduced into the GPD1 locus of the genome through multi-fragment homologous recombination and CRISPR-Cas9-mediated selection marker deletion technology to obtain the final mutant strain.

[0015] Thirdly, the present invention provides the application of the high-glycerol-producing Saccharomyces cerevisiae mutant strain in fermented food brewing, particularly in wine brewing.

[0016] Preferably, the application involves inoculating the mutant strain into grape juice for fermentation to increase the glycerol content in the final product and enhance the roundness and fullness of the wine.

[0017] Preferably, for white wine fermentation, the fermentation temperature is controlled at 16-18℃; for red wine, the fermentation temperature is controlled at 20-25℃.

[0018] The beneficial effects of this invention are as follows:

[0019] 1. This invention, through a combination of rational design and in vivo validation, successfully obtained a single key mutation site, I184L, which has been experimentally verified to significantly increase glycerol production. The target is clear and the effect is remarkable.

[0020] 2. The constructed mutant strain achieved traceless editing in industrial-grade diploid yeast, contains no exogenous resistance genes, has high biosafety, and is more suitable for the food industry.

[0021] 3. This strain significantly increases glycerol production (by approximately 45%-51% compared to the original strain) without affecting normal cell growth, fermentation rate, or aroma spectrum, perfectly balancing the two major requirements of "high yield" and "high quality".

[0022] Biological Preservation Instructions

[0023] Saccharomyces cerevisiae G410 was deposited on November 19, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 39061. Attached Figure Description

[0024] Figure 1 A diagram of the glycerol metabolism pathway in Saccharomyces cerevisiae;

[0025] Figure 2 Sequence alignment diagram of glycerol-3-phosphate dehydrogenase;

[0026] Figure 3 Molecular docking diagram of glycerol-3-phosphate dehydrogenase;

[0027] Figure 4 Diagram of molecular strategy for in situ site-directed mutagenesis in haploid yeast;

[0028] Figure 5 Diagram of molecular strategy for in situ site-directed mutagenesis in diploid yeast;

[0029] Figure 6 Plasmid mapping;

[0030] Figure 7 Wine fermentation rate curve;

[0031] Figure 8 A comparison chart of glycerol production in wine. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments and accompanying drawings, so that those skilled in the art can better understand and implement the present invention. However, the listed embodiments are not intended to limit the present invention. Unless otherwise specified, the methods used in the following embodiments are conventional methods.

[0033] The starting strain used in this invention, Saccharomyces cerevisiae G410, was deposited at the China General Microbiological Culture Collection Center on November 19, 2025, with the accession number CGMCC No. 39061.

[0034] Example 1: Rational Design and In vivo Validation of Optimal Mutation Site for Glycerol-3-phosphate Dehydrogenase (GPD1)

[0035] 1. Rational design for predicting potential mutation sites

[0036] Using Chimera 1.18, the *Saccharomyces cerevisiae* GPD1 protein (PDB ID: 4FGW) was molecularly docked with its substrate dihydroacetone phosphate (DHAP) and coenzyme NADH to determine the enzyme's active pocket and key interacting residues. Simultaneously, the *Saccharomyces cerevisiae* GPD1 sequence was compared with homologous enzymes from the high-glycerol-producing *Candida glycerinogenes*. Combining the docking and alignment results, five potential mutation sites that could affect catalytic efficiency were identified: F73Y, I184L, T186P, R132K, and T305S.

[0037] 2. In vivo functional validation using haploid yeast

[0038] 2.1 Strains and Primers

[0039] Five single-point mutant strains were obtained from the laboratory haploid Saccharomyces cerevisiae strain FY1679-01B (MATa; ura3-52) via homologous recombination. The primer sequences used for gene manipulation are shown in the table below:

[0040] Table 1 Primer Sequences

[0041]

[0042] 2.2 Construction of GPD1 gene knockout strain (ΔGPD1::kanMX)

[0043] Using the genome of strain FY1679-01B as a template, fragments flanking the GPD1 gene were amplified using primers GPD1-shang-F / R and GPD1-xia-F / R, respectively. Using plasmid pUMRI-21 containing the kanMX selection marker as a template, the kanMX fragment with homologous arms to the GPD1 locus was amplified using primer GPD1-KanMX-F / R. This fragment was transformed into FY1679-01B competent cells using the lithium acetate transformation method and plated on YPD plates containing G418 (200 μg / mL) for selection. Colony PCR confirmed the successful replacement of the GPD1 open reading frame with the kanMX marker, yielding the knockout strain FY-ΔGPD1.

[0044] The steps for preparing competent yeast cells required for the transformation process are as follows:

[0045] (1) Pick the yeast colonies to be transformed from the plate and inoculate them into 2 mL of YPD liquid medium, and incubate overnight at 30°C and 220 rpm.

[0046] (2) Transfer the activated yeast seed culture to a 250 mL shake flask containing 50 mL of freshly prepared YPD liquid medium (initial OD). 600 (Approximately 0.2-0.3), incubate at 30℃ and 220 rpm for 3-4 hours to allow OD... 600 It reached around 0.8;

[0047] (3) Transfer the bacterial culture to a 50 mL centrifuge tube, centrifuge at 8,000 rpm for 3 min at room temperature, discard the supernatant, add 30 mL of sterile water to resuspend the cells, centrifuge at 8,000 rpm for 3 min at room temperature, and discard the supernatant. Add 1 mL of freshly prepared 1×TE / 1×LiAc, mix thoroughly, and the resulting yeast competent cells are ready. Yeast competent cells should be freshly prepared before each transformation to ensure the transformation rate.

[0048] 2.3 Construction of GPD1 gene complement strains carrying different point mutations

[0049] Using the constructed plasmid pUMRI-GPD1 as a template, five point mutations (F73Y, I184L, T186P, R132K, and T305S) were introduced via overlap extension PCR to obtain mutated GPD1 gene fragments. Each fragment was transformed into FY-ΔGPD1 competent cells and plated on YPD plates. After colonies grew, they were replicated to YPD plates containing G418 using the replication method. Colonies that grew on YPD plates but not on YPD+G418 plates were identified as positive transformants with the kanMX marker replaced by the mutated GPD1 gene. Sequencing confirmed the mutations were correct, yielding five single-point mutant strains: 01B-GPD1 (F37Y), 01B-GPD1 (I184L), 01B-GPD1 (T186P), 01B-GPD1 (R132K), and 01B-GPD1 (T305S).

[0050] 2.4 Fermentation Experiment and Glycerol Yield Determination

[0051] Wild-type FY1679-01B and five mutant strains were inoculated into YPD liquid medium containing 80 g / L glucose and fermented statically at 24°C for 96 hours. After fermentation, the supernatant was collected, and the glycerol concentration was determined by high-performance liquid chromatography (HPLC).

[0052] 3. Results

[0053] Table 2. Glycerol yield of each strain in YPD medium

[0054]

[0055] 4. Conclusion

[0056] Experimental results showed that among the five rationally designed predicted mutation sites, the I184L mutation had the most significant effect on increasing glycerol yield, increasing it by 97.09% compared to the wild type, and no significant inhibition of strain growth was observed. Therefore, I184L was identified as the optimal mutation site for constructing a high-yield industrial glycerol strain. Mutations at other sites had limited or no significant effect on increasing glycerol yield.

[0057] Example 2 Construction of a high-glycerol-producing yeast mutant strain G410-M184

[0058] In industrial diploid Saccharomyces cerevisiae G410, the optimal mutation site I184L determined in Example 1 was precisely and seamlessly introduced into the GPD1 locus of the genome through multi-fragment homologous recombination and CRISPR-Cas9-mediated marker deletion technology, thereby constructing a high-yield glycerol-producing engineered strain that can be directly used for fermentation production.

[0059] 1.1 Strains and Primers

[0060] Using laboratory diploid Saccharomyces cerevisiae G410 as the starting strain, the G410-M184 strain was obtained by site-directed mutagenesis at position 184 of GPD1. The open reading frame of the GPD1 gene is shown in SEQ ID NO: 35. Mutating codon 184 of this gene from ATC to CTC yielded the GPD1 mutant gene carrying the I184L point mutation, the sequence of which is shown in SEQ ID NO: 36 and confirmed by sequencing. The primer sequences used for gene manipulation are shown in the table below:

[0061] Table 3 Primer Sequences

[0062]

[0063] 1.2 Assembly and transformation of donor DNA fragments

[0064] The following three fragments were assembled sequentially into a complete donor DNA fragment using overlap extension PCR or multi-fragment recombination technology:

[0065] ① Fragment A (upstream homologous arm): Amplified using G410 genomic DNA as a template with primers GPD1-shang-F / R(wu).

[0066] ②Fragment B (selection marker): Using plasmid pCAMBIA-35s-GFP as a template, the HPT gene was amplified using primer GPD1-HPT-F / R(wu).

[0067] ③ Fragment C (overlapping upper homologous arm, mutant gene and downstream homologous arm): Using the constructed plasmid pUMRI-GPD1-184 as a template, primers GPD1-shang-NF(wu) and GPD1-xia-R(wu) were used to amplify part of the upper homologous arm, the GPD1 ORF carrying the I184L point mutation and the downstream homologous arm.

[0068] The fragment was transformed into G410 competent cells using the lithium acetate conversion method (specific method as shown in 2.2 of Example 1), plated on YPD plates containing hygromycin B (concentration 200 μg / mL), and cultured at 30°C for 2-3 days.

[0069] 1.3 Screening and Validation of Intermediate Strain G410-Δgpd1::hpt-GPD1(I184L)

[0070] Hygromycin B resistant transformants were selected and colony PCR was performed using primers GPD1-shang-w-yz-F and GPD1-xia-w-yz-R for verification. The PCR products were analyzed by agarose gel electrophoresis; correct transformants should amplify a fragment larger than the wild-type band, and the band should be unique. The correctly verified strain was named G410-Δgpd1::hpt-GPD1(I184L).

[0071] 1.4 CRISPR-Cas9-mediated seamless deletion of screening markers

[0072] A gRNA sequence targeting the coding region of the HPT gene was designed and cloned into a yeast CRISPR plasmid containing a Cas9 expression unit, constructing the plasmid Cas-HPT-gRNA. The plasmid Cas-HPT-gRNA was transformed into competent cells of the intermediate strain G410-Δgpd1::hpt-GPD1(I184L) and plated on YPD plates containing G418 (hygromycin B) to screen for transformants containing the CRISPR plasmid. Subsequently, the transformants were replicated to YPD plates and YPD+hygromycin B plates using a replication method. Colonies that grew well on YPD plates but not on YPD+hygromycin B plates indicated that their HPT gene had been precisely deleted by CRISPR-Cas9 through homologous recombination repair via the remaining homologous arms on both sides. The selected positive clones were continuously passaged in antibiotic-free YPD liquid medium to lose the Cas-HPT-gRNA plasmid. Finally, strains sensitive to both G418 and hygromycin B were screened on YPD and YPD+G418 plates by the photocopying method.

[0073] 2. Results and Validation

[0074] The final candidate strains were validated as follows:

[0075] ①Phenological verification: The growth of this strain on YPD, YPD+G418 and YPD+hygromycin B plates was as expected (growth only on YPD).

[0076] ② Genotype verification: PCR amplification and sequencing were performed using primers GPD1-yz-F / R. The sequencing chromatogram clearly showed that codon 184 of the GPD1 gene was successfully mutated from ATC (encoding isoleucine I) to CTC (encoding leucine L), and no HPT gene sequence or CRISPR plasmid residue was found.

[0077] ③ Final strain naming: The strain that has been verified to be correct is named Saccharomyces cerevisiae G410-M184.

[0078] Example 3: Fermentation experiment of high-glycerol-producing engineered Saccharomyces cerevisiae G410-M184 in winemaking

[0079] 1. Materials and Methods

[0080] To further determine the glycerol-producing capacity of the yeast mutant strain G410-M184 in grape juice, this experiment used red and white grape juice for fermentation. The red grapes were Marselan grapes harvested in 2025 from the eastern foothills of the Helan Mountains in Ningxia, with a reducing sugar content of 223 g / L, a pH of 3.44, and a total titratable acidity of 4.76 g / L. The white grapes were Chardonnay grapes harvested in 2025 from the eastern foothills of the Helan Mountains in Ningxia, with a reducing sugar content of 256 g / L, a pH of 3.90, and a total titratable acidity of 3.63 g / L.

[0081] The control groups in this experiment were wild yeast strain G410, commercial Saccharomyces cerevisiae D254, and natural fermentation of grape juice. Wild yeast strain G410, yeast mutant strain G410-M184, and commercial Saccharomyces cerevisiae D254 were inoculated into 1 L of grape juice, respectively. Red grape juice was fermented statically at 24°C, and white grape juice was fermented statically at 18°C. Alcoholic fermentation was considered complete when the densitometer reading was less than 0.995. Samples taken at the end of fermentation were used to determine the sugar, acid, and volatile aroma compounds.

[0082] Determination of the content of major metabolites in fermentation broth:

[0083] After filtration of the fermentation broth (PES, 0.22 μm), the major fermentation metabolites were analyzed using HPLC 1200 (Agilent Technologies, Inc. Palo Alto, CA). The ion-exchange column was an HPX-87H Aminex ion-exchange column (300 × 7.8 mm, Bio-Rad Laboratories, USA), the mobile phase was 5 mM H₂SO₄ solution, isocratic elution was performed, and the flow rate was 0.6 mL / min.

[0084] Glucose, fructose, ethanol, and glycerol were determined using a refractive index detector (RID, G1362A, Agilent Technologies, USA) with an injection volume of 20 μL, a column temperature of 45 °C, and an analysis time of 30 min. Organic acids (tartaric acid, malic acid, citric acid, lactic acid, succinic acid, and acetic acid) were determined using a photodiode array detector (DAD, G1315D, Agilent Technologies, USA) with an injection volume of 10 μL, a column temperature of 60 °C, and an analysis time of 25 min.

[0085] Determination of volatile aroma compounds such as esters, higher alcohols, and organic acids in the fermentation broth: The types and contents of various volatile aroma compounds in the fermented wine obtained above were detected using an Agilent 6890 gas chromatograph (GC) and an Agilent 5975 mass spectrometer (MS) (Agilent, USA). Specific conditions were as follows: a capillary column (HP-INNOWAX Polyethylene Glycol 60m × 0.25mm × 0.25μm, J&W Scientific, USA) was used with high-purity helium as the carrier gas at a flow rate of 1 mL / min; headspace solid-phase microextraction was performed using splitless mode, inserted into the GC inlet at 250℃, with thermal desorption for 25 min. The column oven temperature program was: 40℃ for 5 min, then increased to 200℃ at a rate of 3℃ / min and held for 2 min. The mass spectrometer interface temperature is 280℃, the ion source temperature is 230℃, the ionization mode is EI, the ion energy is 70 eV, and the mass scan range is 20-350 m / z.

[0086] Add 5 mL of fermentation sample to a 15 mL sample vial, along with 1 g NaCl and 10 µL of internal standard (4-methyl-2-pentanol). Seal the vial immediately with a PTFE septum cap and equilibrate at 180 r / min for 30 min at a constant temperature of 40 °C. Once the gas-liquid phase aroma compounds in the vial have reached equilibrium, insert an activated or thermally desorbed polydimethylsiloxane / carbon sieve / divinylbenzene (PDMS / CAR / DVB) extraction head into the headspace of the vial. Extract with stirring at 40 °C for 30 min to achieve gas-solid and gas-liquid equilibrium. Then, insert the extraction head into the GC-MS inlet and perform thermal desorption at 250 °C for 8 min, followed by splitless injection.

[0087] Qualitative and quantitative analysis of aroma compounds: For substances with existing standards, qualitative analysis was performed using full-ion mass spectrometry (Scan) based on retention time, retention index, and mass spectrometry information under the same chromatographic conditions established in this experiment. Quantitative analysis was then performed using a standard curve in a simulated wine solution (the synthetic wine solution was an aqueous solution of 2 g / L glucose, 7 g / L tartaric acid, and 12% alcohol, with pH adjusted to 3.3 using NaOH. The mixed aroma standards were prepared in 15 gradients). For substances without standards, semi-qualitative analysis was performed using the retention index of the compound under similar chromatographic conditions reported in the literature and comparison results with the NIST 11 standard library (NIST Chemistry WebBook).

[0088] 2. Results and Analysis of Core Metabolites

[0089] Table 4. Content of major metabolites in each experimental group after alcoholic fermentation of white wine.

[0090]

[0091] Note: Data in the table (mean ± standard deviation); significance analysis of variance was performed using one-way ANOVA and Tukey HSD post-hoc test. Different letters after the content of the same compound indicate significant differences between different treatment groups (p<0.05).

[0092] Table 5. Content of major metabolites in each experimental group of rosé wine after alcoholic fermentation.

[0093]

[0094] Note: Data in the table (mean ± standard deviation); significance analysis of variance was performed using one-way ANOVA and Tukey HSD post-hoc test. Different letters after the content of the same compound indicate significant differences between different treatment groups (p<0.05).

[0095] As shown in Tables 4 and 5, the basic physicochemical indicators of each fermentation experimental group are all within the national standard control range and meet the requirements of GB / T 15037-2006 for various indicators of wine. The mutant strain G410-M184 constructed in this invention exhibits significant and consistent excellent characteristics in wine fermentation.

[0096] In Chardonnay white wine, G410-M184 achieved a glycerol yield of 9.66 g / L, a 45.0% increase compared to its originating strain G410 (6.66 g / L); in Marselan rosé wine, the glycerol yield reached 11.85 g / L, a 51.1% increase compared to G410 (7.84 g / L). This increase was statistically significant (p<0.05), fully achieving the invention's high glycerol yield target.

[0097] Corresponding to the increased glycerol production, the ethanol production of G410-M184 showed a slight but significant decrease in both experiments (white wine: approximately 0.7% vol; rosé wine: approximately 0.6% vol). This result is consistent with expectations from metabolic engineering, demonstrating that the I184L mutation effectively enhances glycerol-3-phosphate dehydrogenase activity, diverting more carbon from the main ethanol synthesis pathway to the glycerol synthesis pathway. In both experiments, the residual sugar (glucose + fructose) content of G410-M184 was comparable to or lower than that of the control group, indicating thorough fermentation and unimpaired fermentation kinetics. The content of major fixed acids (such as tartaric acid, malic acid, and citric acid) was not significantly different from the control group or was within the ideal range. Although acetic acid increased in both white and rosé wines, it remained at an excellent level. This indicates that the mutation did not lead to the accumulation of undesirable volatile acid byproducts, which is crucial for ensuring the sensory quality of the wine.

[0098] The above metabolite data demonstrate that the yeast mutant strain G410-M184 can specifically and efficiently increase the glycerol content in wine without affecting the basic fermentation process, and its regulation of the organic acid spectrum is beneficial to the roundness and harmony of the wine's taste.

[0099] 3. Results and Analysis of Volatile Aroma Compounds

[0100] Table 6. Aroma compound content of white wine in each experimental group after alcoholic fermentation.

[0101]

[0102]

[0103] Note: Data in the table (mean ± standard deviation); significance analysis of variance was performed using one-way ANOVA and Tukey HSD post-hoc test. Different letters after the content of the same compound indicate significant differences between different treatment groups (p<0.05).

[0104] Table 7. Aroma compound content of rosé wines in each experimental group after alcoholic fermentation.

[0105]

[0106]

[0107] Note: Data in the table (mean ± standard deviation); significance analysis of variance was performed using one-way ANOVA and Tukey HSD post-hoc test. Different letters after the content of the same compound indicate significant differences between different treatment groups (p<0.05).

[0108] A comprehensive analysis of the volatile aroma compounds in Tables 6 and 7 shows that the yeast mutant strain G410-M184 significantly increased glycerol production while having a positive and controllable impact on the aroma profile of wine. Its aroma characteristics were not deteriorated by the metabolic pathway modification, but rather exhibited a new, balanced metabolic homeostasis.

[0109] Esters are the main contributors to fruit aromas in wine. Data shows that ethyl fatty acid esters remain dominant in total content. In white wine, the total ethyl fatty acid ester content of G410-M184 (20410.1 μg / L) was not significantly different from that of the commercial strain D254 (19049.8 μg / L), but significantly higher than that of the originating strain G410 (15744.0 μg / L). Among these, ethyl hexanoate and ethyl octanoate, which impart aromas of apple and pineapple, were present in the highest amounts. Although the contents of isoamyl acetate (imparting banana aromas) and phenylethyl acetate (imparting rose and honey aromas) decreased, the contents of long-chain ethyl fatty acids such as ethyl laurate and ethyl myristate, which contribute to the fullness of wine, significantly increased in white wine. This change may lead to a shift in fruit aroma types from a rich banana aroma to a more complex, mellow fruit and waxy aroma, rather than a loss in the total amount of aromas.

[0110] Higher alcohols are fundamental to the structure and complexity of wine. There was no significant difference in the total higher alcohol content among the three strains. In both types of wine, the total higher alcohol content of G410-M184 was not statistically significantly different from the two control groups (p>0.05), indicating that no abnormal higher alcohol metabolic stress was generated during fermentation. Specifically, in the white wine, the G410-M184 fermented wine had significantly higher levels of citronellol (with rose and citrus aromas) and 4-terpene alcohol (with woody and floral aromas) than the control group. In the rosé wine, the levels of citronellol and 4-terpene alcohol were also significantly higher than or equal to the optimal control group. These terpenes are important indicators for evaluating the aroma quality of wine varieties, and an increase in their content has a positive impact on quality.

[0111] Considering the total amount of all aroma substances, including esters, higher alcohols, and fatty acids, there was no order of magnitude difference between G410-M184 and the control groups, indicating that its aroma metabolism was in a balanced state overall, without any abnormal accumulation or deficiency of any type of substance.

[0112] In conclusion, Example 3 demonstrates that strain G410-M184 can significantly increase the glycerol content of wine, optimize the acid profile, and maintain excellent aroma characteristics, making it perfectly suitable for the production of high-quality wines.

[0113] The G410-M184 strain constructed in this invention significantly increases glycerol production (45%-51%) while successfully maintaining the overall balance and excellent quality of the wine's aroma system. Although its aroma characteristics differ distinguishably from the starting strain (such as the redistribution of some esters), this difference is reflected in the optimization of specific beneficial components (such as some fatty acid ethyl esters and terpenes), and key undesirable substances (volatile acids) are controlled. Therefore, this mutant strain can impart a rounder mouthfeel to wine while fully ensuring and potentially optimizing its flavor complexity, possessing extremely high industrial application value.

[0114] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A high-glycerol-producing engineered strain of *Saccharomyces cerevisiae*, characterized in that, Its glycerol-3-phosphate dehydrogenase (GPD1) contains the I184L point mutation.

2. The *Saccharomyces cerevisiae* mutant strain according to claim 1, characterized in that, The strain is an industrial brewing yeast, classified as Saccharomyces cerevisiae G410-M184.

3. A method for constructing a *Saccharomyces cerevisiae* mutant strain as described in claim 1 or 2, characterized in that, Includes the following steps: (1) By molecular docking and multiple sequence alignment, potential high-activity mutation sites of glycerol-3-phosphate dehydrogenase were rationally designed and screened, and in vivo functional verification was performed using a haploid yeast model. The optimal mutation site was determined to be I184L. (2) In diploid industrial brewing yeast, the GPD1 gene containing the I184L mutation and the selection marker gene are integrated into the GPD1 locus of the genome through homologous recombination; (3) Using CRISPR-Cas9 system-mediated gene editing, the selection marker gene is deleted to achieve scarless editing and obtain the mutant strain.

4. The construction method according to claim 3, characterized in that, The homologous recombination described in step (2) is a multi-segment homologous recombination, and the donor fragment includes at least: an upstream homologous arm, a selection marker gene, and a GPD1 gene carrying the I184L mutation and a downstream homologous arm.

5. The application of the mutant strain of Saccharomyces cerevisiae as described in claim 1 or 2 in the brewing of fermented foods.

6. The application according to claim 5, characterized in that, The application is in winemaking; preferably, the mutant strain is inoculated into grape juice for fermentation.

7. The application according to claim 6, characterized in that, The fermentation conditions are as follows: the fermentation temperature for white wine is controlled at 16-18℃, and the fermentation temperature for red wine is controlled at 20-25℃.

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