Preparation method and application of saccharomyces cerevisiae strain with high flocculation efficiency

Abstract

The invention provides a preparation method and application of a saccharomyces cerevisiae strain with high flocculation efficiency, a specific gene is knocked out and transferred in by using a homologous recombination method through a gene engineering technology to modify a saccharomyces cerevisiae BY4741 strain, and the metabolic pathway and related performance of yeast are accurately regulated and controlled. The method comprises the following specific steps: knocking out the SNF11 gene of the saccharomyces cerevisiae BY4741 strain, restoring the SNF11-SS18 (QPGY) gene, and constructing to obtain the saccharomyces cerevisiae strain with high flocculation efficiency, so that the efficient aggregation of yeast cells in a fermentation tank is realized, the cell density is improved, the fermentation period is shortened, expensive equipment such as a centrifugal machine is avoided, the energy consumption is reduced, and the production cost is reduced. Therefore, the fermentation efficiency is remarkably improved, the production cost is reduced, and powerful technical support is provided for sustainable development of the alcoholic fermentation industry.

Description

Technical Field

[0001] This invention belongs to the field of alcoholic fermentation, specifically relating to a method for preparing a brewing yeast strain with high flocculation efficiency and its application. Background Technology

[0002] In the technological development of the alcohol fermentation industry, the effective utilization of yeast has always been a core element, with its performance and application methods playing a crucial role in the efficiency, cost, and product quality of the entire production process. However, current mainstream technologies still face many bottlenecks that urgently need to be addressed.

[0003] Due to the physical characteristics of yeast cells—small size and high water content—free yeast fermentation is difficult to maintain in the fermenter within a continuous fermentation system, and they are easily lost with the flow of the fermentation mash. This directly results in a consistently low effective density of yeast cells within the fermenter, severely hindering the fermentation reaction rate, significantly reducing production efficiency, and failing to meet the demands of modern industrial production for high efficiency and speed. In the yeast cell separation stage after fermentation, the dispersed state of free yeast necessitates expensive centrifugation equipment, consuming substantial energy to separate the yeast from the fermentation broth, increasing energy costs and further reducing profit margins for enterprises.

[0004] Traditional immobilized yeast technology uses adsorption, encapsulation, and cross-linking to fix yeast cells, which has successfully increased the density of yeast cells in fermenters to a certain extent, thereby shortening fermentation time and having a positive impact on industrial development. However, its high dependence on various carrier materials, such as polymers and gels, has led to problems such as high procurement costs, difficult storage, complex application operations, and the potential for impurities to interfere with yeast metabolism, affecting fermentation quality and yield stability.

[0005] Protoplast fusion technology is highly complex and technically challenging, significantly increasing research and development time and costs. Furthermore, the complex genetic background obtained through this technology can lead to decreased genetic stability during subsequent subculturing and fermentation due to gene recombination and loss, resulting in fluctuations in the strain's self-flocculation and fermentation performance, affecting production stability and product quality consistency. Simultaneously, protoplast fusion is a relatively random gene combination method, making it difficult to precisely regulate specific genes to optimize strain performance. Targeted enhancement or weakening of certain key gene functions is challenging to achieve, failing to fully meet specific production needs.

[0006] Based on the flocculation performance of existing yeast strains, most naturally occurring or conventionally bred yeast strains perform poorly in this area and cannot achieve efficient automatic flocculation. After fermentation, yeast cells are difficult to separate quickly and effectively from the fermentation broth, making post-processing extremely complex and cumbersome. Furthermore, under long-term industrial fermentation applications, yeast strains exhibit poor genetic stability and are prone to genetic mutations. These mutations lead to fluctuations in yeast fermentation performance, making it difficult to maintain stable alcohol yield and quality, thus affecting production management and product quality control.

[0007] The existence of these technological bottlenecks severely restricts the sustainable development of the alcohol fermentation industry, necessitating breakthroughs through technological innovation. This invention, based on in-depth gene function research, involves genetic engineering modification of the *Saccharomyces cerevisiae* BY4741. SNF11, a key component of the chromatin remodeling complex SWI / SNF, has been shown in studies to have no effect on the growth of *Saccharomyces cerevisiae* upon knockout, providing a safe basis for subsequent gene manipulation. SS18, a homolog of SNF11 in humans, possesses a unique QPGY domain at its C-terminus, which can influence normal physiological functions in mammals through a phase separation mechanism. Based on this, this invention utilizes genetic engineering techniques to perform gene knockout and transfer operations on *Saccharomyces cerevisiae* BY4741, precisely regulating the yeast's metabolic pathways and related properties. This avoids interference with yeast metabolism caused by impurities introduced by the carrier material, thereby optimizing the overall performance of the strain during alcohol fermentation. This ensures that the flocculation and fermentation performance of the yeast strain remains stable throughout continuous fermentation and multiple passages, meeting the alcohol fermentation industry's demand for high-quality, high-efficiency production. Summary of the Invention

[0008] Technical Problem to be Solved: To address the aforementioned technical problems, this invention provides a method for preparing and applying a high-flocculation-efficiency *Saccharomyces cerevisiae* strain. Using genetic engineering techniques, *Saccharomyces cerevisiae* BY4741 is precisely modified by knocking out the SNF11 gene and then restoring the SNF11-SS18 (QPGY) gene, thus constructing a *Saccharomyces cerevisiae* strain with high flocculation efficiency. The high-flocculation-efficiency yeast strain prepared by this invention exhibits significant autoflocculation characteristics, high genetic stability, and excellent fermentation performance, playing a significant role in improving production efficiency, reducing costs, and enhancing product quality stability in the field of alcoholic fermentation.

[0009] Technical solution: A high-flocculation-efficiency Saccharomyces cerevisiae strain is constructed by using Saccharomyces cerevisiae strain BY4741 as the starting strain and SNF11 as the target gene. The SNF11 gene is first knocked out and then the SNF11-SS18 (QPGY) gene is restored. The nucleotide sequence of the high-flocculation-efficiency Saccharomyces cerevisiae strain is shown in SEQ ID NO:1.

[0010] The method for preparing the above-mentioned high-flocculation-efficiency Saccharomyces cerevisiae strain includes the following steps:

[0011] S1. SNF11 gene knockout: Design and construct a DNA fragment with a marker gene for homologous recombination, introduce the constructed DNA fragment for homologous recombination into Saccharomyces cerevisiae cells, spread it on a medium containing selection markers for screening, and verify the selected colonies by PCR to obtain yeast cells with the SNF11 gene knocked out.

[0012] S2. SNF11-SS18 gene restoration: The SNF11-SS18 gene and its promoter region were cloned into the pRS305 plasmid. The restoration plasmid containing the SNF11-SS18 gene was linearized by single enzyme digestion. The linearized restoration plasmid was introduced into yeast cells in which the SNF11 gene had been successfully knocked out. Using the selection gene carried in the restoration plasmid, the cells were screened on a culture medium with the corresponding selection conditions. The selected yeast cells were verified by PCR to obtain yeast cells with SNF11-SS18 integration.

[0013] Preferably, the DNA fragment used for homologous recombination in step S1 is a sequence containing the Ura selection marker gene in the middle and sequences homologous to the upstream and downstream of the SNF11 gene at both ends.

[0014] Preferably, the nucleotide sequences of the primers required for PCR verification in step S1 are SNF11p-F1-GTGGAGAACAGACTTCCCATAAAAGC and Ura-R1-GAACGTATGGGAGGAAGAGAGAAGAAG.

[0015] Preferably, the size of the promoter subregion in step S2 is 1000 bp.

[0016] Preferably, the enzyme used for single enzyme digestion in step S2 is Eco81I.

[0017] Preferably, the gene screened in step S2 is Leu.

[0018] Preferably, the nucleotide sequences of the primers required for PCR verification in step S2 are SNF11-SS18(QPGY)-F1-TTTCCTTTTTTCCCAGAAAAATGAGCAGTGAAATTG and SNF11-SS18(QPGY)-R1-CGACGGTATCGATAAGCTTTTACTGCTGGTAATTTC.

[0019] Application of the above-mentioned high-flocculation-efficiency Saccharomyces cerevisiae strains in alcoholic fermentation.

[0020] The inoculum size of the above-mentioned high-flocculation-efficiency Saccharomyces cerevisiae strains in alcoholic fermentation is 1~6.5×10⁻⁶. 5 CFU / mL.

[0021] Beneficial effects:

[0022] 1. The high flocculation efficiency brewing yeast strain prepared by this invention exhibits significant autoflocculation characteristics. The flocculated yeast particles can achieve free sedimentation in a short time. Compared with traditional natural or conventionally selected yeast strains, its flocculation efficiency is greatly improved, which enables the yeast cells to maintain a high density in the fermenter, thereby accelerating the fermentation speed, significantly shortening the alcohol fermentation time, greatly shortening the separation time, and improving production efficiency.

[0023] 2. The high flocculation efficiency Saccharomyces cerevisiae strain prepared by this invention greatly improves the stability of fermentation quality. Precise gene regulation avoids interference with yeast metabolism caused by impurities introduced by carrier materials, making the fermentation process more stable and the alcohol yield and quality easier to control.

[0024] 3. The high-flocculation-efficiency Saccharomyces cerevisiae strain prepared by this invention does not require complex separation equipment. Due to its highly efficient flocculation characteristics, there is no need to use expensive equipment such as centrifuges to separate yeast cells from the fermentation broth after alcoholic fermentation. This not only reduces equipment investment costs but also lowers energy consumption costs, thereby reducing production costs in multiple ways.

[0025] 4. The high flocculation efficiency Saccharomyces cerevisiae strain prepared by this invention has strong genetic stability. By using genetic engineering techniques to perform precise gene knockout and gene reversion operations on Saccharomyces cerevisiae BY4741, the constructed strain has a clearer and more stable genetic background. During continuous fermentation and multiple generations, the flocculation and fermentation performance of the yeast strain remains stable, providing a reliable strain guarantee for industrial production. Attached Figure Description

[0026] Figure 1 The images show morphological comparisons of the BY4741 strain transformed with SNF11-SS18 (QPGY) under a conventional optical microscope (Example 1), the wild-type BY4741 strain (Comparative Example 1), and the BY4741 strain with SNF11 knocked out (Comparative Example 2).

[0027] Figure 2 The images show the microscopic morphological comparison of the BY4741 strain transformed with SNF11-SS18 (QPGY) (Example 1), the wild-type BY4741 strain (Comparative Example 1), and the BY4741 strain with SNF11 knocked out (Comparative Example 2) under scanning electron microscopy (SEM).

[0028] Figure 3The growth of BY4741 strain transformed with SNF11-SS18 (QPGY) (Example 1), wild-type BY4741 strain (Comparative Example 1), BY4741 strain with SNF11 knocked out (Comparative Example 2), and BY4741 complement strain Re-SNF11 transformed with RS305-SNF11 after SNF11 gene knockout (Comparative Example 5) under various culture conditions;

[0029] Figure 4 For BY4741 strain transformed with SNF11-SS18 (QPGY) (Example 1), wild-type BY4741 strain (Comparative Example 1), and BY4741 strain with SNF11 knockout (Comparative Example 2), under the condition of consistent initial cell density, the OD within 100 min was... 600 Value changes;

[0030] Figure 5 The polyacrylamide gel electrophoresis (SDS-PAGE) images show the expression of GFP fusion protein in the BY4741 strain transformed with SNF11-SS18 (QPGY) (Example 1), the wild-type BY4741 strain (Comparative Example 1), and the BY4741 strain with SNF11 knocked out (Comparative Example 2).

[0031] Figure 6 Laser confocal microscopy localization diagrams showing the differences in intracellular distribution of GFP fusion protein in BY4741 strain transformed with SNF11-SS18 (QPGY) (Example 1), wild-type BY4741 strain (Comparative Example 1), and BY4741 strain with SNF11 knocked out (Comparative Example 2). Detailed Implementation

[0032] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments:

[0033] The *Saccharomyces cerevisiae* strain BY4741 used in the following examples and comparative examples was preserved in the inventor's laboratory; *Saccharomyces cerevisiae* CECA was purchased from Angel Yeast Co., Ltd., with a viable count of 10-1. 8 CFU / g.

[0034] Example 1

[0035] A method for preparing a Saccharomyces cerevisiae strain with high flocculation efficiency includes the following steps:

[0036] S1. Design of knockout primers and amplification of sequences: Based on the genome sequence information of Saccharomyces cerevisiae, specific knockout primers were designed for the SNF11 gene. The 5' end of the upstream primer corresponds to the 39 bp sequence before the SNF11 start codon, and the 20 bp at the 3' end corresponds to the sequence of the universal primer T1 (Table 1); the 5' end of the downstream primer corresponds to the 39 bp sequence after the SNF11 stop codon (including the stop codon), and the 20 bp at the 3' end corresponds to the LRS sequence (Table 1); using plasmid pUG72 as a template, PCR amplification was performed, and DNA fragments carrying the Ura marker gene were screened. The primer sequences are listed in Table 2, the PCR sequences in Table 3, and the PCR reaction conditions in Table 4.

[0037] S2. Yeast cell transformation: Saccharomyces cerevisiae BY4741 strain was inoculated into 6 mL of YPD liquid medium and cultured at 30°C and 200 rpm on a shaker until OD... 600 Collect bacterial cells by centrifugation at 3000 rpm for 1 min at a pH between 0.4 and 0.6; wash yeast cells with 1 mL ddH2O and centrifuge at 4000 rpm for 1 min; treat yeast cells twice with 1 mL LiTE and centrifuge at 4000 rpm for 1 min; resuspend yeast cells in 320 μL LiTE / PEG, add the DNA fragment with the Ura marker gene obtained in S1 and 20 μL of treated ssDNA (denatured at 100℃ for 10 min, annealed on ice for 2 min), mix well; shake at 30℃ and 250 rpm for 30 min; heat shock at 42℃ for 40 min, then incubate on ice for 1 min; centrifuge at 6000 rpm for 1 min and discard the supernatant; wash the precipitate once with 1 mL TE; resuspend yeast cells in 200 μL ddH2O and plate onto SD-Ura selection plates; incubate the plates upside down at 30℃ for 2–4 days, and pick single colonies for PCR identification;

[0038] S3. Colony PCR identification: Primer sequences are listed in Table 2, PCR product series are listed in Table 3, and PCR reaction conditions are listed in Table 4. The upstream primer corresponds to the promoter region 1000 bp before the SNF11 start codon, and the downstream primer corresponds to the selection marker gene Ura. The PCR product is controlled to be within 1000 bp. The PCR product is detected by agarose gel electrophoresis to screen out yeast strains with successful SNF11 gene knockout.

[0039] S4. SNF11-SS18 (QPGY) gene reversion:

[0040] (1) Construction of the recovery plasmid: The SNF11-SS18 (QPGY) fusion gene and its 1000bp promoter region were constructed into the RS305 plasmid vector using molecular cloning technology. A suitable single enzyme digestion site was found in the 1000bp promoter region to linearize the plasmid.

[0041] (2) Transformation and screening: The linearized reversion plasmid was transformed into SNF11 gene knockout Saccharomyces cerevisiae cells, and the transformation method was the same as in step S2. The selection gene Leu carried by the reversion plasmid was used for screening. The screening plates were cultured at 30℃ for 2-4 days, and single colonies were picked for PCR identification.

[0042] (3) Colony PCR identification: Primer sequences are listed in Table 5, PCR sequences are listed in Table 6, PCR reaction conditions are listed in Table 7, and PCR products are detected by agarose gel electrophoresis to verify whether the SNF11-SS18 (QPGY) gene has been successfully integrated into the yeast genome.

[0043] (4) Western Blot: Extract total protein from yeast cells and use Western Blot to detect the expression of SNF11-SS18 (QPGY) protein to ensure successful gene reversion and normal expression.

[0044] Table 1. Sequences required for primer design

[0045]

[0046] Table 2 Primer sequences for identifying SNF11 gene knockout

[0047]

[0048] Table 3. PCR system for identifying SNF11 gene knockout

[0049]

[0050] Table 4. PCR reaction conditions for identifying SNF11 gene knockout

[0051] Table 5 Primer sequences for identifying SNF11-SS18 (QPGY) integration

[0052]

[0053] Table 6. PCR systems for identifying SNF11-SS18 (QPGY) integration

[0054]

[0055] Table 7. PCR reaction conditions for identifying SNF11-SS18 (QPGY) integration

[0056]

[0057] Example 2

[0058] This embodiment describes the application of the high flocculation efficiency Saccharomyces cerevisiae strain prepared in Example 1 in alcoholic fermentation, including the following steps:

[0059] S1. Strain activation and culture: The high flocculation efficiency Saccharomyces cerevisiae strain prepared in Example 1 was inoculated into 5 mL of YPD liquid medium and cultured in a shaker at 30 °C and 200 rpm for 20 h.

[0060] S2. Simulated Fermentation Performance Test: The simulated grape juice culture medium contained the following components: 100 g / L glucose, 100 g / L fructose, 0.3 g / L citric acid, 3 g / L tartaric acid, 3 g / L malic acid, 0.2 g / L sodium chloride, and 2 g / L ammonium sulfate. The pH was adjusted to 5.0 with 0.5 mol / L sodium hydroxide, and the mixture was filtered to remove bacteria. The activated, highly flocculating Saccharomyces cerevisiae was then introduced at a rate of 6 × 10⁻⁶. 5 Inoculate the simulated grape juice medium with an inoculum of CFU / mL and culture anaerobically at 30°C.

[0061] S3. Observation and detection: At 96 h of culture, 5 mL of fermentation broth was taken from the culture system and transferred to a 10 mL centrifuge tube. The mixture was allowed to stand at room temperature for 15 min. The clarity of the fermentation broth was observed, and the flocculation characteristics, degree of flocculation, total acid, reducing sugar, alcohol content and pH value were detected.

[0062] Example 3

[0063] The difference between this embodiment and Embodiment 2 is that the inoculum size of the high-flocculation-efficiency Saccharomyces cerevisiae in this embodiment is 4 × 10⁻⁶. 5 CFU / mL.

[0064] Example 4

[0065] The difference between this embodiment and Embodiment 2 is that the inoculum size of the high-flocculation-efficiency Saccharomyces cerevisiae in this embodiment is 2 × 10⁻⁶. 5 CFU / mL.

[0066] To further illustrate the technical effects of the present invention, a comparative example is also provided, as follows:

[0067] Comparative Example 1

[0068] The difference between this comparative example and Example 2 is that the Saccharomyces cerevisiae strain used in this comparative example is wild-type Saccharomyces cerevisiae BY4741, and the inoculum size is 2 × 10⁻⁶. 5 CFU / mL.

[0069] Comparative Example 2

[0070] The difference between this comparative example and Example 2 is that the Saccharomyces cerevisiae strain used in this comparative example is Saccharomyces cerevisiae BY4741 with the SNF11 gene knocked out, and the inoculum size is 2 × 10⁻⁶. 5 CFU / mL.

[0071] Comparative Example 3

[0072] The difference between this comparative example and Example 2 is that the Saccharomyces cerevisiae strain in this comparative example is Saccharomyces cerevisiae BY4741 transformed with the RS305 empty vector, and the inoculum size is 2 × 10⁻⁶. 5 CFU / mL.

[0073] Comparative Example 4

[0074] The difference between this comparative example and Example 2 is that the Saccharomyces cerevisiae strain in this comparative example is Saccharomyces cerevisiae BY4741, which was transformed into the RS305 empty vector after the SNF11 gene was knocked out, and the inoculum size is 2×10⁻⁶. 5 CFU / mL.

[0075] Comparative Example 5

[0076] The difference between this comparative example and Example 2 is that the Saccharomyces cerevisiae strain in this comparative example is the BY4741 complement strain Re-SNF11, which was transformed with RS305-SNF11 after the SNF11 gene was knocked out, and the inoculum size is 2 × 10⁻⁶. 5 CFU / mL.

[0077] Comparative Example 6

[0078] The difference between this comparative example and Example 2 is that the Saccharomyces cerevisiae strain used in this comparative example is industrial-grade high-flocculation Saccharomyces cerevisiae CECA, and the inoculum size is 2 × 10⁻⁶. 5 CFU / mL, fermentation temperature 25℃.

[0079] Indicator Test:

[0080] 1. Autoflocculation Characteristics: Transfer 5 mL of yeast fermentation broth to a 10 mL centrifuge tube and let it stand at room temperature for 15 min. Observe the clarification of the fermentation broth. If the upper layer of the fermentation broth gradually becomes clear and obvious yeast cell precipitate appears at the bottom, it indicates that the strain has autoflocculation characteristics. At the same time, observe the distribution of yeast cells under a microscope using a hemocytometer. If the cells are aggregated rather than uniformly dispersed, it further proves its flocculation characteristics. Record the time when obvious flocculation is first observed as an important indicator of the strain's autoflocculation characteristics.

[0081] 2. Degree of flocculation:

[0082] (1) Microscopic observation of cell aggregation morphology: At different time points after culture, 10 μL of fermentation broth was dropped onto a glass slide, covered with a coverslip, and observed under an optical microscope (200x magnification). Image acquisition software was used to record cell aggregation morphology and measure the size and number of cell aggregates. For measuring the size of cell aggregates, multiple fields of view were selected, and the long axis and short axis lengths of 10 aggregates were randomly measured in each field of view to calculate the average size. For the number of aggregates, the number of aggregates in each field of view was directly counted, and the average value was taken after statistical analysis of multiple fields of view.

[0083] (2) Turbidity change measurement: Take 3 mL of fermentation liquid into a cuvette, use the uninoculated fermentation medium as a blank control, and use a turbidimeter to measure the turbidity of the fermentation liquid at a wavelength of 600 nm. Measure once every 15 min. Plot the turbidity change curve with time as the abscissa and turbidity value as the ordinate. Record the time required for the turbidity to drop to 50% of the initial turbidity. The shorter the time, the higher the degree of flocculation.

[0084] (3) Recording of specific flocculation effect time: The specific flocculation effect is set as a 60% reduction in the turbidity of the fermentation broth. From the start of cultivation, the turbidity change is continuously monitored and the time required to achieve the specific flocculation effect is recorded. If the effect is not achieved within 96 hours of cultivation, it is recorded as "not up to standard". At the same time, the degree of flocculation of the strain is comprehensively evaluated by combining the cell aggregation morphology and turbidity change curve observed under a microscope.

[0085] 3. Foam Production Performance: Yeast cells were inoculated into 5 mL of YPD liquid medium and cultured on a shaker at 30℃ and 200 rpm for 20 h. 2 mL of the bacterial suspension was centrifuged at 12000 rpm for 1 min to collect the cells. This process was repeated once. The cells were resuspended in 200 μL of YPD liquid medium and then inoculated into 10 mL of YPD liquid medium. The culture was then incubated at 30℃ for 24 h. Foam height was measured every 4 h, and the highest foam height within 24 h was recorded.

[0086] 4. After fermentation, the total acid, reducing sugar, alcohol content and pH in the fermentation broth were determined according to GB / T 15038-2006.

[0087] Figure 1These are morphological comparison images of the BY4741 strain transformed with SNF11-SS18 (QPGY) (Example 1), the wild-type BY4741 strain (Comparative Example 1), and the BY4741 strain with SNF11 knockout (Comparative Example 2) under a regular optical microscope. Under this field of view, the wild-type BY4741 strain (Comparative Example 1) and the BY4741 strain with SNF11 knockout (Comparative Example 2) cells are evenly distributed in a dispersed state, with clear individual cell outlines, intact spherical or ellipsoidal cell morphology, and no obvious aggregation. However, the BY4741 strain transformed with SNF11-SS18 (QPGY) (Example 1) exhibits significant cell flocculation characteristics, with a large number of yeast cells agglomerating to form cell clusters of varying sizes. Some cell clusters are tightly connected, exhibiting a filamentous or network structure. Furthermore, the cell wall thickness and overall cell outline of the two strains were basically similar, further confirming that the SNF11-SS18 (QPGY) fusion protein had no significant inhibitory effect on yeast cell growth, but could induce specific flocculation by changing cell surface properties.

[0088] Figure 2 These are scanning electron microscope (SEM) images comparing the microscopic morphology of BY4741 strain SNF11-SS18 (QPGY) (Example 1), wild-type BY4741 strain (Comparative Example 1), and BY4741 strain with SNF11 knockout (Comparative Example 2). High-magnification observation reveals that all three strains maintain a typical spherical or ellipsoidal cell shape, with no significant differences in cell wall surface texture, thickness, or budding structure. The outer cell membranes are continuous and smooth, without any observed wrinkles, damage, or abnormal protrusions. Furthermore, the amount and distribution of secretions on the cell surface of the three strains are also similar, indicating that the introduction of SNF11-SS18 (QPGY) did not significantly affect the microstructure and surface properties of yeast cells. This further corroborates the experimental conclusion that the fusion protein specifically regulates yeast flocculation properties without interfering with normal cell growth and morphological maintenance.

[0089] Figure 3 The growth of BY4741 strain with SNF11-SS18 (QPGY) (Example 1), wild-type BY4741 strain (Comparative Example 1), and BY4741 strain with SNF11 knockout (Comparative Example 2) under various culture conditions is shown. The BY4741 strain with SNF11-SS18 (QPGY) (Example 1) exhibited the same stress resistance as the wild-type strain under various environmental stresses, and did not gain any additional environmental adaptation advantage due to gene transfer.

[0090] Figure 4The changes in OD values ​​within 100 min were compared between the SNF11-SS18 (QPGY) BY4741 strain (Example 1), the wild-type BY4741 strain (Comparative Example 1), and the SNF11 knockout BY4741 strain (Comparative Example 2) under the condition of consistent initial cell density. The cell density of the SNF11-SS18 (QPGY) BY4741 strain (Example 1) decreased significantly within 100 min, while the cell densities of the wild-type BY4741 strain (Comparative Example 1) and the SNF11 knockout BY4741 strain (Comparative Example 2) remained relatively stable. Throughout the growth cycle, the trend of optical density (OD) values ​​in yeast cells showed no statistically significant difference compared to the wild-type strain, indicating that the fusion protein did not significantly affect the proliferation activity and biomass accumulation of yeast cells.

[0091] Figure 5 To facilitate observation, GFP tags were added to the C-terminus of the target genes in the BY4741 strain with SNF11-SS18 (QPGY) (Example 1), the wild-type BY4741 strain (Comparative Example 1), and the BY4741 strain with SNF11 knockout (Comparative Example 2). The expression of different GFP fusion proteins (GFP, SNF11-GFP, SNF11-QPGY-GFP) was detected using α-GFP antibody, with α-β-tubulin antibody detection of β-tubulin expression serving as a control. It can be seen that the target proteins of each strain were successfully expressed in the experimental system. Furthermore, the signal intensity of the β-tubulin band was relatively consistent in each lane, indicating uniform sample loading and comparable experiments.

[0092] Figure 6 After culturing different GFP-labeled strains in the logarithmic growth phase, cells were stained with Hoechst dye and imaged. The figure shows representative images, with merged channels including GFP, Hoechst, and brightfield images, scale bar (2 μm). The results showed that the distribution of different fusion proteins in cells differed: GFP in the BY4741 strain with SNF11 knockout (Comparative Example 2) was diffusely distributed; SNF11-GFP protein in the wild-type BY4741 strain (Comparative Example 1) was diffusely distributed in cells; and SNF11-SS18 (QPGY) in the BY4741 strain (Example 1) formed discrete punctate aggregates in the cell nucleus.

[0093] Table 8 Performance Indicators of Simulated Fermentation by Yeast

[0094]

[0095] Table 8 shows the simulated fermentation performance results of Examples 2-4 and Comparative Examples 1-6. There were no significant differences in any of the indicators among the wild-type BY4741 strain (Comparative Example 1), the BY4741 strain with SNF11 knockout (Comparative Example 2), the BY4741 strain transformed with the RS305 empty vector (Comparative Example 3), the BY4741 strain with SNF11 gene knockout and transformed with the RS305 empty vector (Comparative Example 4), and the BY4741 complement strain Re-SNF11 with SNF11 gene knockout and transformed with RS305-SNF11 (Comparative Example 5). This indicates that neither knocking out the SNF11 gene nor transforming with the RS305 vector changes the fermentation performance of the strains. Compared with Comparative Examples 1-5, the SNF11-SS18 (QPGY) BY4741 strains (Examples 2-4) exhibited high flocculation and low foam production. The rapid settling characteristic resulting from high flocculation can shorten the clarification time, while the low foam production characteristic can reduce production losses. Compared with other comparative examples, Examples 2-4 exhibited superior fermentation capabilities, with key indicators significantly outperforming the other control groups. Specifically, at the end of fermentation, Examples 2-4 had the lowest residual reducing sugar and pH values ​​among all groups, while achieving the highest total acid content and alcohol content. Although commercially available yeast (Comparative Example 6) also exhibited high flocculation and low foaming properties, its fermentation capabilities were weaker than those of Examples 2-4.

[0096] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A Saccharomyces cerevisiae strain with high flocculation efficiency, characterized in that: The high-flocculation-efficiency Saccharomyces cerevisiae strain is constructed by taking Saccharomyces cerevisiae strain BY4741 as a starting strain, taking SNF11 as a target gene, knocking out the SNF11 gene, and then restoring the SNF11-SS18 gene.

2. The high flocculation efficiency Saccharomyces cerevisiae strain according to claim 1, characterized in that: The nucleotide sequence encoding the high-flocculation-efficiency Saccharomyces cerevisiae strain is shown as SEQ ID NO:

1.

3. The method for preparing a Saccharomyces cerevisiae strain with high flocculation efficiency according to claim 1 or 2, characterized in that, The method comprises the following steps: S1. SNF11 gene knockout: a DNA fragment for homologous recombination with a marker gene is designed and constructed, the constructed DNA fragment for homologous recombination is introduced into Saccharomyces cerevisiae cells, and is coated on a culture medium containing a screening marker for screening, and the screened colonies are verified by PCR to obtain Saccharomyces cerevisiae cells with the SNF11 gene knocked out; S2. SNF11-SS18 gene restoration: the SNF11-SS18 gene and its promoter region are cloned into a pRS305 plasmid, the restoration plasmid containing the SNF11-SS18 gene is single-enzyme-digested to linearize the plasmid, the linearized restoration plasmid is introduced into the Saccharomyces cerevisiae cells with the SNF11 gene successfully knocked out, and the screening gene carried in the restoration plasmid is used to screen on a culture medium containing a corresponding screening condition, and the screened Saccharomyces cerevisiae cells are verified by PCR to obtain Saccharomyces cerevisiae cells with the SNF11-SS18 gene integrated.

4. The method of claim 3, wherein: The DNA fragment for homologous recombination in step S1 is a Ura screening marker in the middle and sequences homologous to the upper and lower SNF11 genes at both ends.

5. The method of claim 3, wherein: The nucleotide sequences of the primers required for PCR verification in step S1 are SNF11p-F1-GTGGAGAACAGACTTCCCATAAAAGC and Ura-R1-GAACGTATGGGAGGAAGAGAAGAAG.

6. The method of claim 3, wherein: In step S2, the size of the fragment of the promoter region is 1000 bp; the enzyme used for single-enzyme digestion is Eco81 I; and the screening gene is Leu.

7. The method of claim 3, wherein: The nucleotide sequences of the primers required for PCR verification in step S2 are SNF11-SS18-F1-TTTCCTTTTTTCCCAGAAAAATGAGCAGTGAAATTG and SNF11-SS18-R1-CGACGGTATCGATAAGCTTTTACTGCTGGTAATTTC.

8. Application of the high-flocculation-efficiency Saccharomyces cerevisiae strain of claim 1 or 2 in alcohol fermentation.

9. Use according to claim 8, characterized in that: The inoculation amount of the high flocculation efficiency Saccharomyces cerevisiae strain in alcohol fermentation is 1-6.5x10 5 CFU / mL.

Images