A pdc1 gene mutant strain resistant to 2-phenylethanol and application thereof in brewing of maixiang type liquor

Abstract

This invention relates to the fields of microbial genetic engineering and brewing technology, and provides a PDC1 gene mutant strain resistant to 2-phenylethanol and its application in the brewing of wheat-aroma baijiu. The mutant strain is *Saccharomyces cerevisiae* PDC1_473 (HM 03), with the preservation number CCTCC M20252996. The PDC1 gene of this strain has a base mutation from T to C at position 473. When this mutant strain is applied to the solid-state fermentation of wheat-aroma baijiu, it becomes the absolutely dominant fungal community, significantly altering the microbial community structure of the fermentation system, inhibiting the growth of various conditionally pathogenic and spoilage bacteria, strengthening the fermentation environment centered on *Lactobacillus*, and significantly enhancing the rose, citrus, grassy, ​​fatty, and caramel aromas of the baijiu. It imparts a pleasant, rich ester aroma and complex aroma to the baijiu, increasing its fullness, and has broad application potential in the brewing of wheat-aroma baijiu.

Description

Technical Field

[0001] This invention relates to the fields of microbial genetic engineering and brewing technology, specifically to a PDC1 gene mutant strain tolerant to 2-phenylethanol and its application in the brewing of wheat-flavored baijiu. Background Technology

[0002] 2-Phenylethanol (2-PE) is one of the important flavor compounds in baijiu (Chinese liquor), contributing a pleasant rose aroma. Its content has a significant impact on the quality and flavor profile of baijiu. Currently, the formation of flavor compounds in baijiu depends on a complex microbial community, and traditional processes struggle to target and increase the yield of specific beneficial flavor components (such as 2-phenylethanol).

[0003] As one of the core functional bacteria in baijiu brewing, *Saccharomyces cerevisiae* has limited tolerance to end products, which may inhibit its metabolic activity and affect fermentation efficiency and flavor stability. Pyruvate decarboxylase (encoding gene PDC1) is a key enzyme in the yeast glycolysis pathway, and PDC1 gene modification may affect yeast stress response and metabolic flux. Therefore, in view of the above situation, there is an urgent need to provide a PDC1 gene mutant strain tolerant to 2-phenylethanol and its application in baijiu brewing to overcome the shortcomings in current practical applications. Summary of the Invention

[0004] The purpose of this invention is to provide a PDC1 gene mutant strain resistant to 2-phenylethanol and its application in the brewing of wheat-flavored baijiu, aiming to solve the problems in the background art mentioned above.

[0005] The present invention is achieved by a PDC1 gene mutant strain resistant to 2-phenylethanol, wherein the base T at position 473 of the PDC1 gene of the strain is mutated to C, and its nucleic acid sequence is shown in SEQ ID NO:1.

[0006] As a further aspect of the present invention: the strain is *Saccharomyces cerevisiae* PDC1_473 (HM 03), classified as *Saccharomyces cerevisiae* PDC1_473, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M20252996, located at Wuhan University, No. 229 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on December 24, 2025.

[0007] The present invention also provides a method for constructing a PDC1 gene mutant strain resistant to 2-phenylethanol as described above, comprising the following steps:

[0008] Step 1: Construction of recombinant pML104 plasmid:

[0009] The pML104 plasmid was extracted and linearized pML104 plasmid backbone was obtained by digestion with restriction endonucleases BclI and Smil.

[0010] Oligostranded gRNAs targeting the mutation site at position 473 of the PDC1 gene were designed, and double-stranded gRNAs were obtained through hybridization.

[0011] Double-stranded gRNA was ligated to the linearized pML104 plasmid backbone, transformed into E. coli JM110 competent cells, positive transformants were screened and sequenced to verify, and recombinant pML104-pdc1gRNA plasmid was obtained.

[0012] Step 2, Donor fragment amplification:

[0013] Using the genome of Saccharomyces cerevisiae CEN.PK113-5D as a template, the upstream and downstream homologous arms of the PDC1 gene were amplified by PCR, and then the upstream and downstream homologous arms were fused by fusion PCR to obtain the Donor fragment containing the T mutation to C at the 473rd base of the PDC1 gene.

[0014] Step 3, conversion of brewing yeast:

[0015] The recombinant pML104-pdc1gRNA plasmid and the Donor fragment were co-transformed into Saccharomyces cerevisiae CEN.PK113-5D competent cells. Positive transformants were picked on SD-URA3 solid plates, and genomic DNA was extracted and sequenced to verify the results, thus obtaining a PDC1 gene mutant strain.

[0016] As a further aspect of the present invention: in step 1, the hybridization reaction conditions are as follows:

[0017] Incubate at 95°C for 6 minutes, then repeat 70 cycles from 95°C to 25°C, with each cycle decreasing by 1°C and each cycle lasting 1 minute.

[0018] The present invention also provides the application of the 2-phenylethanol-tolerant PDC1 gene mutant strain described above in the brewing of wheat-flavored baijiu.

[0019] As a further aspect of the present invention, the seed liquid of the PDC1 gene mutant strain that is tolerant to 2-phenylethanol is added to a traditional solid-state fermentation system for baijiu (Chinese liquor) fermentation.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] The PDC1 gene mutant strain (Saccharomyces cerevisiae PDC1_473) constructed in this invention exhibits significant tolerance to 2-phenylethanol. The wild-type strain can hardly grow in YEPD solid plates containing 3.0 g / L 2-PE, while this mutant strain can grow in YEPD solid plates containing both 3.0 g / L and 3.5 g / L 2-PE. Furthermore, when this mutant strain ferments with L-phenylalanine as a substrate for 48 h, the 2-phenylethanol concentration reaches 2.64 g / L, which is 22% higher than the 2.16 g / L of the wild-type strain, achieving a targeted increase in 2-phenylethanol yield.

[0022] When this mutant strain was applied to solid-state fermentation of baijiu, it became the dominant fungal species, exhibiting strong environmental adaptability and competitiveness. At the same time, it significantly altered the bacterial community structure of the fermentation system, effectively inhibiting the growth of various conditionally pathogenic and spoilage bacteria such as Enterobacter, Klebsiella, and Escherichia-Shigella, without significantly disrupting the basic fermentation pattern dominated by Lactobacillus and Weissella. This ensured the normal acidity process of fermentation and was beneficial to the formation of baijiu flavor (ester balance).

[0023] The application of this mutant strain significantly enhanced the rose, citrus, grassy, ​​fatty, and caramel aromas of baijiu, giving it a pleasant, rich ester aroma and complex fragrance, increasing its fullness, and effectively improving its quality. Attached Figure Description

[0024] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0025] Figure 1 This is an agarose gel electrophoresis image of the PCR amplification products.

[0026] Figure 2 This is a schematic diagram showing the growth of Saccharomyces cerevisiae CEN.PK113-5D (wild-type strain) and Saccharomyces cerevisiae PDC1_473 strain on YEPD solid plates with 3.0 g / L and 3.5 g / L 2-PE.

[0027] Figure 3 Schematic diagram showing the 2-PE concentration produced by wild-type Saccharomyces cerevisiae and PDC1_473 strain.

[0028] Figure 4This is a schematic diagram showing the changes in fungal (A) and bacterial (B) communities in a mash sample fermented for 30 days.

[0029] Figure 5 A schematic diagram illustrating the differences in flavor compounds in distilled baijiu after fermentation with added brewing yeast strain PDC1_473. Detailed Implementation

[0030] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0032] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art will understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0033] The present invention will be further explained below with reference to specific embodiments.

[0034] Example 1: Construction of a PDC1 gene mutant strain tolerant to 2-phenylethanol:

[0035] First, the gRNA and the enzyme-digested pML104 plasmid were ligated to obtain the recombinant pML104 plasmid (pML104-pdc1gRNA). This recombinant plasmid targets and recognizes the base sequence near the PDC1 gene mutation site. Then, PCR amplification was performed using the Saccharomyces cerevisiae CEN.PK113-5D genome as a template to obtain upstream and downstream homologous arms to PDC1. Subsequently, fusion PCR was used to fuse the upstream and downstream homologous arms to obtain the Donor fragment, as shown in the figure. Figure 1 As shown (in) Figure 1 In the diagram, Figure A shows the upstream (1, 2) and downstream (3, 4) homologous arms of PDC1 amplified by PCR; Figure B shows the fusion PCR product. Each Donor fragment was successfully recovered from the gel and sequenced to verify its success.

[0036] Finally, the Donor fragment and the corresponding recombinant pML104 plasmid were co-transformed into *Saccharomyces cerevisiae* CEN.PK113-5D, and positive transformants were picked from SD-URA3 solid plates. Genomic DNA was then extracted from the positive transformants and *Saccharomyces cerevisiae* CEN.PK113-5D, and PCR amplification was performed using validation primers. The amplified DNA fragments were recovered and sent to Sangon Biotech Co., Ltd. for sequencing verification, resulting in a successful mutant strain named *Saccharomyces cerevisiae* PDC1_473 (HM 03), deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M20252996, located at Wuhan University, No. 229 Bayi Road, Wuchang District, Wuhan City, Hebei Province, on December 24, 2025. The viability of this culture was tested by the collection center on December 31, 2025, and the result was positive.

[0037] Example 2: 2-PE tolerance test of PDC1 gene mutant strains:

[0038] To investigate the effect of PDC1 gene mutation on the tolerance of Saccharomyces cerevisiae to 2-PE stress, Saccharomyces cerevisiae CEN.PK113-5D (wild-type strain) and Saccharomyces cerevisiae PDC1_473 strain were grown on solid plates containing different concentrations of 2-PE.

[0039] The results are as follows Figure 2 As shown, the wild-type strain could not grow at all on YEPD solid plates containing 3.0 g / L 2-PE, while the mutant strain could grow on YEPD solid plates containing both 3.0 g / L and 3.5 g / L 2-PE.

[0040] The above experimental results indicate that the PDC1 gene mutation significantly improves the 2-PE tolerance of Saccharomyces cerevisiae.

[0041] Example 3: Detection of the ability of PDC1 gene mutant strains to synthesize 2-PE:

[0042] Using L-phenylalanine as a substrate, the synthesis of 2-PE in *Saccharomyces cerevisiae* strain PDC1_473 was detected, and the results are as follows: Figure 3 As shown. After 48 h of fermentation, the 2-PE concentration of the mutant strain was 2.64 g / L, which was 22% higher than that of the wild-type strain (2.16 g / L).

[0043] In conclusion, the PDC1 gene mutation (the base T at position 473 is changed to C) not only enhances the stress tolerance to 2-PE, but also enhances the ability to synthesize 2-PE.

[0044] The nucleic acid sequence of the PDC1 gene of the strain is shown in SEQ ID NO:1.

[0045] SEQ ID NO:1:

[0046]

[0047] Example 4: Application of PDC1 gene mutant strain in the brewing of wheat-flavored baijiu:

[0048] To analyze the effects of exogenous addition of Saccharomyces cerevisiae strain PDC1_473 on bacteria and fungi in the mash, this application added Saccharomyces cerevisiae strain PDC1_473 culture medium at 10%-20% of the weight of the starter culture, with a cell concentration of 1×10⁻⁶. 7 cfu / mL. Then, 16 s and 18 s amplicon analysis and body composition analysis were performed on the mash samples after 30 days of fermentation.

[0049] The results showed that the addition of strain PDC1_473 significantly increased the content of β-phenylethanol, a typical floral aroma compound, in the wine. Meanwhile, the content of active pentanol and furfural significantly decreased, which greatly reduced the potential off-flavors (Table 1).

[0050] Table 1. Wine Components

[0051] Element control group Add PDC1_473 group Ethyl acetate 553.09±5.20 540.81±1.08 methanol 85.68±0.28 84.01±0.26 β-Phenylephethanol <![CDATA[23.43±0.02 a ]]> <![CDATA[42.69±0.03 b ]]> furfural <![CDATA[317.85±1.75 a ]]> <![CDATA[155.136±0.09 b ]]> Ethyl lactate <![CDATA[1057.04±1.76 a ]]> <![CDATA[400.46±0.88 b ]]> Ethyl hexanoate 12.08±0.68 16.05±1.26 Ethyl butyrate 5.65±0.08 3.65±0.08 Active pentanol <![CDATA[510.66±3.27 a ]]> <![CDATA[115.25±10.22 b <!-- 4 -->]]> Isoamyl alcohol <![CDATA[86.10±2.39 a ]]> <![CDATA[115.25±4.50 b ]]> Isobutanol 228.98±0.87 292.07±0.19 sec-butanol 0.72±0.03 0.55±0.01 n-Propanol 70.97±0.44 97.86±0.28

[0052] When exogenous Saccharomyces cerevisiae strain PDC1_473 was added, this mutant strain became the absolutely dominant fungal community after fermentation. Figure 4 A). This indicates that the strain possesses strong environmental adaptability and competitive ability. In the experimental group, in addition to the growth of Saccharomyces, several yeast genera related to brewing showed consistent growth, such as Cyberlindnera-Candida_clade, Pichia, Saccharomycopsis, and Wickerhamomyces-Candida_clade.

[0053] The addition of the brewing yeast strain PDC1_473 significantly altered the bacterial community structure of the fermentation system, effectively inhibiting the growth of various conditionally pathogenic and spoilage bacteria, such as Enterobacter, Klebsiella, and Escherichia-Shigella. Furthermore, the addition of the PDC1_473 mutant strain did not significantly disrupt the basic fermentation pattern dominated by Lactobacillus and Weissella, ensuring the normal acidity process of fermentation, which is crucial for the formation of baijiu flavor (ester balance).

[0054] To further analyze the effect of exogenous addition of *Saccharomyces cerevisiae* strain PDC1_473 on the flavor of post-fermentation distilled baijiu, this application analyzed the volatile metabolites in a 52% vol sample. For the differentially expressed metabolites and annotated sensory flavor characteristics identified based on screening criteria in the two comparison groups, the top 10 sensory flavors with the highest number of annotations were used to construct radar and Sankey diagrams. The results showed that the addition of *Saccharomyces cerevisiae* strain PDC1_473 significantly enhanced the rose, citrus, grassy, ​​fatty, and caramel aromas in the baijiu, imparting a pleasant, rich ester aroma and complex fragrance, and increasing its fullness.

[0055] The specific method for constructing a PDC1 gene mutant strain tolerant to 2-phenylethanol is as follows:

[0056] 1. Construction of recombinant strains:

[0057] This application uses CRISPR / Cas9 gene editing technology to mutate PDC1 based on the pML104 plasmid. The pML104 plasmid contains both a Cas9 expression cassette and an sgRNA expression cassette.

[0058] Construction of recombinant pML104 plasmid:

[0059] pML104 plasmid extraction: E. coli JM110 glycerol tubes containing pML104 plasmid (stored at -80℃) were inoculated into LB liquid medium containing 100 μg / mL ampicillin sodium and cultured overnight at 37℃ and 200 rpm.

[0060] The activated bacterial culture was transferred to LB liquid medium containing 100 μg / mL ampicillin sodium and incubated at 37°C and 200 rpm for 12–16 h. The pML104 plasmid was extracted according to the FastPure Plasmid Mini Kit instructions and its concentration was detected using Nanodrop.

[0061] Construction of linearized pML104 plasmid: 1 μg of pML104 was incubated with restriction endonucleases BclI (Thermo Scientific FastDigest BclI) and Smil (Thermo Scientific FastDigest SmiI) at 37°C for 20 min to obtain the linearized pML104 plasmid backbone. The linearized plasmid backbone was recovered from the gel and used for sgRNA cloning. The enzyme digestion reaction system for pML104 plasmid is shown in Table 2.

[0062] Table 2. Reaction system for enzyme digestion of pML104 plasmid

[0063] name volume BclI 1 μL Smil 1 μL pML104 16 μL Green Buffer 2 μL

[0064] Oligomeric gRNA hybridization: Based on the mutation information of the PDC1 (473T>C) gene, gRNAs (pdc1gRNA-f / pdc1gRNA-r) were designed approximately 60 bp upstream and downstream of the mutation site and sent to Wuhan Bioengineering Co., Ltd. for synthesis. The synthesized oligomeric gRNAs were then configured according to the system in Table 3 for hybridization.

[0065] Table 3 Hybridization reaction system

[0066] name volume pdc1gRNA-f (10 μM) 6 μL pdc1gRNA-r (10 μM) 6 μL 10×T4 Buffer 2 μL pure water 6 μL

[0067] The hybridization conditions are as follows:

[0068] Incubate at 95°C for 6 minutes, then repeat 70 cycles from 95°C to 25°C, with each cycle decreasing by 1°C and each cycle lasting 1 minute.

[0069] Double-stranded gRNA and pdc1gRNA were obtained and targeted to the PDC1 gene. The obtained pdc1gRNA and linearized pML104 were configured according to the system in Table 4 and then ligated.

[0070] Table 4. T4 DNA ligase reaction system

[0071] name volume T4 ligase 1 μL 10×T4 Buffer 2 μL Fragment (pdc1gRNA) 0.5 μL Vector (linearized pML104) 4 μL pure water 12.5 μL

[0072] The system was reacted overnight at 16°C to obtain the recombinant plasmid pML104-pdc1gRNA.

[0073] Transformation of recombinant pML104 plasmid:

[0074] (1) Take 200 μL of E. coli JM110 competent cells and place them on ice to thaw. Add more than 10 μL of recombinant mixture (pML104-pdc1gRNA), mix gently and let stand on ice for 30 min.

[0075] (2) Heat shock in a 42℃ water bath for 90 s, then quickly place on ice for 3 min.

[0076] (3) Add 900 μL of LB medium and culture at 37°C and 200 rpm for 45 min to revive the bacteria and express the antibiotic resistance marker gene encoded by the plasmid.

[0077] (4) Centrifuge at 5000 r / min for 2 min, retain 100 μL of supernatant, mix well and spread on LB plates containing ampicillin sodium. Incubate upside down in a 37℃ incubator for 12-18 h, pick positive transformants, enrich and extract recombinant plasmids, and use T3 primers for sequencing to verify whether the recombination was successful.

[0078] Amplification of the Donor fragment:

[0079] Using *Saccharomyces cerevisiae* CEN.PK113-5D as a template, upstream and downstream homologous arms were amplified by PCR using primer pair PDC1-UF / PDC1-UR. Then, an equimolar mixture of the upstream and downstream arms was used as a template, and amplification was performed using primer pair PDC1-UF / PDC1-DR to obtain a fusion fragment, which is the Donor fragment of the PDC1 gene mutation. This Donor fragment contains a mutation at position 473 of the PDC1 gene (T becomes C).

[0080] Saccharomyces cerevisiae conversion:

[0081] (1) Activate Saccharomyces cerevisiae CEN.PK113-5D, culture overnight at 30℃ and 200 r / min, then transfer to 50 mL YEPD liquid medium. When OD 600 Once the bacterial culture reaches a viscosity of 0.6-0.8, collect the bacterial solution, centrifuge at 5000 rpm for 5 min at 4℃, and collect the bacterial cells.

[0082] (2) Wash twice with ice-cold sterile water and centrifuge at 4°C and 5000 rpm for 5 min.

[0083] (3) Add 1 mL of sterile 0.1 M LiAc to resuspend the bacterial cells, centrifuge at 4℃ and 5000 rpm for 5 min, and discard the supernatant.

[0084] (4) Add 500 μL of 0.1 M LiAc to resuspend the prepared brewer's yeast competent cells and store them on ice.

[0085] (5) Take 50 μL of competent Saccharomyces cerevisiae and add 240 μL of 50% PEG3350, 36 μL of 1 mol / L LiAc, 50 μL of 2 mg / mL salmon sperm single-stranded DNA, 5 μL of recombinant pML104 plasmid and 10 μL of Donor fragment in sequence.

[0086] (6) Whirl violently for 1 minute to mix it evenly.

[0087] (7) Heat the mixed bacterial solution in a water bath at 30°C for 30 min, then heat shock it in a water bath at 42°C for 25 min, centrifuge at 4°C and 5000 rpm for 5 min, and discard the supernatant.

[0088] (8) Add 1 mL of YEPD medium, incubate in a 30℃ water bath for 30 min, and then incubate at 30℃ and 200 rpm for 1 h.

[0089] (9) Centrifuge at 5000 rpm for 5 min, discard the supernatant, add 100 μL of sterile water to resuspend, spread on SD-URA3 plates, and incubate at 30℃ for 2-3 days. Pick positive transformants, extract genomic DNA, and sequence for verification.

[0090] 2. Growth and 2-Phenylene Ethanol Tolerance Detection of Saccharomyces cerevisiae:

[0091] Plate inoculation experiment: Glyceryl-preserved mutant strains and wild-type strains were inoculated into YEPD liquid medium and cultured at 30℃ and 200 rpm for 12 h, respectively. The initial OD was used as the starting point for the inoculation. 600 The inoculum was transferred to YEPD at a rate of 0.5 and cultured at 30°C and 200 rpm. The *Saccharomyces cerevisiae* culture at the logarithmic growth phase was collected by centrifugation, the supernatant was discarded, the cells were resuspended in sterile deionized water, and the OD was measured. 600 and its OD 600 Dilute with sterile water to 0.1. (Based on OD...) 600 Based on 0.1, a 10-fold serial dilution was performed to obtain OD values. 600 0.1×10 -1 10 -2 10 -3 Diluents of different concentrations were prepared. 1 μL or more of each diluent was seeded onto YEPD plates or YEPD plates containing different concentrations of 2-PE and incubated at 30°C.

[0092] In summary, this invention utilizes CRISPR / Cas9 gene editing technology to perform site-directed mutations on the PDC1 gene of Saccharomyces cerevisiae CEN.PK113-5D, successfully constructing a mutant strain of Saccharomyces cerevisiae PDC1_473. This strain significantly improves the stress tolerance to 2-phenylethanol and can increase the yield of 2-phenylethanol in fermentation with L-phenylalanine as a substrate.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A PDC1 gene mutant strain resistant to 2-phenylethanol, characterized in that, The strain has a T-to-C mutation at position 473 of the PDC1 gene, and its nucleic acid sequence is shown in SEQ ID NO:

1.

2. The 2-phenylethanol-tolerant PDC1 gene mutant strain according to claim 1, characterized in that, The strain is *Saccharomyces cerevisiae* PDC1_473, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M20252996, located at Wuhan University, No. 229 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on December 24, 2025.

3. A method for constructing a PDC1 gene mutant strain resistant to 2-phenylethanol as described in claim 1 or 2, characterized in that, Includes the following steps: Step 1: Construction of recombinant pML104 plasmid: The pML104 plasmid was extracted and linearized pML104 plasmid backbone was obtained by digestion with restriction endonucleases BclI and Smil. Oligostranded gRNAs targeting the mutation site at position 473 of the PDC1 gene were designed, and double-stranded gRNAs were obtained through hybridization. Double-stranded gRNA was ligated to the linearized pML104 plasmid backbone, transformed into E. coli JM110 competent cells, positive transformants were screened and sequenced to verify, and recombinant pML104-pdc1gRNA plasmid was obtained. Step 2, Donor fragment amplification: Using the genome of Saccharomyces cerevisiae CEN.PK113-5D as a template, the upstream and downstream homologous arms of the PDC1 gene were amplified by PCR, and then the upstream and downstream homologous arms were fused by fusion PCR to obtain the Donor fragment containing the T mutation to C at the 473rd base of the PDC1 gene. Step 3, conversion of brewing yeast: The recombinant pML104-pdc1gRNA plasmid and the Donor fragment were co-transformed into Saccharomyces cerevisiae CEN.PK113-5D competent cells. Positive transformants were picked on SD-URA3 solid plates, and genomic DNA was extracted and sequenced to verify the results, thus obtaining a PDC1 gene mutant strain.

4. The construction method according to claim 3, characterized in that, In step 1, the hybridization reaction conditions are as follows: Incubate at 95°C for 6 minutes, then repeat 70 cycles from 95°C to 25°C, with each cycle decreasing by 1°C and each cycle lasting 1 minute.

5. The application of a PDC1 gene mutant strain tolerant to 2-phenylethanol as described in claim 1 or 2 in the brewing of wheat-flavored baijiu.

6. The application according to claim 5, characterized in that, The seed culture of the PDC1 gene mutant strain that is tolerant to 2-phenylethanol was added to the solid fermentation system of wheat-flavored baijiu for fermentation.

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