Bacteriostatic saccharification bifunctional strain M:: X-3LysKB317:: XII-1GA-CAS9 as well as construction method and application of bacteriostatic saccharification bifunctional strain M:: X-3LysKB317:: XII-1GA-CAS9

Abstract

The invention discloses a bacteriostatic saccharification bifunctional strain M:: X-3LysKB317:: XII-1GA-CAS9 as well as a construction method and application thereof, and relates to the technical field of biology. The construction method comprises the following steps: integrating a LysKB317 gene and a GA gene into a genome of initial saccharomyces cerevisiae by virtue of a CRISPR / CAS9 gene editing method, and constructing to obtain the bacteriostatic saccharification bifunctional strain M:: X-3LysKB317:: XII-1GA-CAS9, the nucleotide sequence of the LysKB317 gene is as shown in SEQ ID NO. 3; the nucleotide sequence of the GA gene is as shown in SEQ ID NO. 5. The strain can efficiently express peptidoglycan hydrolase and saccharifying enzyme, shows good bacteriostatic activity on infectious microbes such as lactic acid bacteria and the like, and can reduce fermentation pollution without adding antibiotics.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a bifunctional antibacterial and saccharifying strain M::X-3LysKB317::XII-1GA-CAS9, its construction method, and its applications. Background Technology

[0002] The industrial alcohol fermentation process using Saccharomyces cerevisiae involves numerous equipment and pipelines. Anaerobic or facultative anaerobic contaminants can be introduced into the fermentation process through raw materials, water, air, pipelines, and even dead corners in the equipment. Therefore, alcohol fermentation is not a strictly aseptic process. The continuous propagation of yeast and non-aseptic fermentation conditions used in most commercial alcohol fermentation facilities often lead to chronic or unpredictable acute bacterial infections, causing fermentation to terminate. To suppress contamination during industrial alcohol yeast fermentation, acid treatment or the addition of antibiotics is generally used. However, improper acid treatment may reduce yeast activity, and excessively high ion concentrations in the waste liquid increase the difficulty of wastewater treatment and cause significant environmental pollution, making it unsuitable as a long-term method for controlling contamination during industrial alcohol fermentation. Furthermore, the addition of antibiotics such as penicillin, chloramphenicol, and oxytetracycline not only easily leads to bacterial resistance but also leaves antibiotic residues in the fermentation tanks, thus affecting food safety. In addition, novel antibacterial agents such as plant extracts, bioactive peptides, and functional enzymes, which possess antibacterial biological activity, can also effectively inhibit contamination by miscellaneous bacteria without harming the environment. However, the production and purification of bioactive peptides or enzyme preparations are characterized by high costs and low efficiency.

[0003] Industrial bioethanol production primarily uses starch-rich wheat and corn as raw materials. Due to their wide availability and low cost, bioethanol is a promising biological resource. Saccharomyces cerevisiae is an important microorganism in the ethanol fermentation industry, but it cannot directly utilize starch to produce ethanol. Therefore, industrial fermentation has traditionally relied on the addition of exogenous saccharifying enzymes to break down and utilize starch. Methods for producing ethanol from starch include: co-culturing two or more microorganisms capable of hydrolyzing starch and fermenting alcohol in the same fermentation broth; using amylase-containing strains to hydrolyze starch, followed by yeast fermentation to produce alcohol; or first hydrolyzing the starch in the fermentation broth with amylase and saccharifying enzymes, followed by yeast alcoholic fermentation. However, the addition of exogenous enzymes significantly impacts production costs.

[0004] In summary, it is necessary to develop new engineered strains to simplify the production process of bioethanol and reduce production costs. Summary of the Invention

[0005] The purpose of this invention is to provide a dual-function antibacterial and saccharifying bacterial strain M::X-3LysKB317::XII-1GA-CAS9, its construction method, and its applications, to solve the problems existing in the prior art. This strain can efficiently express peptidoglycan hydrolase and saccharifying enzyme, exhibiting good antibacterial activity against lactic acid bacteria and other miscellaneous bacteria, reducing fermentation contamination without the need for antibiotics.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for constructing the antibacterial saccharification bifunctional strain M::X-3LysKB317::XII-1GA-CAS9, comprising the step of integrating the LysKB317 gene and the GA gene into the genome of the original Saccharomyces cerevisiae through CRISPR / CAS9 gene editing, thereby constructing the antibacterial saccharification bifunctional strain M::X-3LysKB317::XII-1GA-CAS9;

[0008] The nucleotide sequence of the LysKB317 gene is shown in SEQ ID NO.3; the nucleotide sequence of the GA gene is shown in SEQ ID NO.5.

[0009] Furthermore, the initial brewing yeast is brewing yeast AMCC 30450, whose accession number at the China Center for Type Culture Collection is CCTCC NO: M 20251982.

[0010] Furthermore, the construction method includes the following steps:

[0011] The LysKB317 gene was integrated into the genome of the original Saccharomyces cerevisiae using the CRISPR / CAS9 gene editing method to obtain the recombinant strain M::X-3LysKB317-CAS9;

[0012] The GA gene was integrated into the genome of the recombinant strain M::X-3LysKB317-CAS9 using the CRISPR / CAS9 gene editing method, resulting in the antibacterial glycosylation bifunctional strain M::X-3LysKB317::XII-1GA-CAS9.

[0013] Furthermore, the method for integrating the LysKB317 gene into the genome of the initial Saccharomyces cerevisiae includes the step of transforming the pH-CAS9 plasmid, the Pscm-X-3N20-V3 plasmid, and the LysKB317 repair fragment into the initial Saccharomyces cerevisiae;

[0014] The nucleotide sequence of the LysKB317 repair fragment is shown in SEQ ID NO.4.

[0015] Furthermore, the Pscm-X-3N20-V3 plasmid is obtained by fusing the guideRNA X-3 sequence to the MCS sequence of the pSCM-N20 plasmid; the nucleotide sequence of the guideRNA X-3 sequence is shown in SEQ ID NO.2.

[0016] Further, the method of integrating the GA gene into the genome of the recombinant strain M::X-3LysKB317-CAS9 includes the steps of transforming the pH-CAS9 plasmid, pscm-XII-1N20-V3 plasmid and GA repair fragment into the recombinant strain M::X-3LysKB317-CAS9;

[0017] The nucleotide sequence of the GA repair fragment is shown in SEQ ID NO.6.

[0018] Furthermore, the Pscm-XII-1N20-V3 plasmid is obtained by fusing the guideRNA XII-1 sequence to the MCS sequence of the pSCM-N20 plasmid; the nucleotide sequence of the guideRNA XII-1 sequence is shown in SEQ ID NO.1.

[0019] The present invention also provides a bifunctional antibacterial and saccharified strain M::X-3LysKB317::XII-1GA-CAS9 constructed according to the above construction method.

[0020] The present invention also provides the application of the above-mentioned antibacterial and saccharifying bifunctional strain M::X-3LysKB317::XII-1GA-CAS9 in the fermentation production of ethanol.

[0021] The present invention also provides a method for producing ethanol by fermentation, comprising the step of producing ethanol by fermentation using the above-mentioned antibacterial and saccharifying bifunctional strain M::X-3LysKB317::XII-1GA-CAS9.

[0022] The present invention discloses the following technical effects:

[0023] This invention utilizes CRISPR / CAS9 gene editing technology to successfully integrate the LysKB317 and GA genes into the genome of *Saccharomyces cerevisiae*, constructing an engineered strain with both antibacterial and saccharification functions, effectively addressing a core challenge in traditional ethanol fermentation. This strain efficiently expresses peptidoglycan hydrolases and saccharifying enzymes, exhibiting excellent antibacterial activity against lactic acid bacteria and other contaminating microorganisms. It reduces fermentation contamination without antibiotics, avoiding drug resistance and food safety risks, while also eliminating the need for acid treatment, thus reducing environmental burden. Regarding saccharification, it efficiently degrades starch without the need for exogenous saccharifying enzymes. In corn mash fermentation experiments, the final alcohol content reached 15.7%, approximately 200% higher than the wild-type strain. Residual sugar utilization was significantly improved, cumulative mass loss was greatly reduced, and production costs were significantly lowered. Furthermore, the strain exhibits excellent tolerance, with ethanol and high sugar tolerance comparable to the wild-type, enhanced acid tolerance, a shortened lag phase of 4 hours, and a more vigorous logarithmic growth phase, meeting the demands of complex industrial fermentation environments. This strain simplifies the ethanol fermentation process and has the advantages of being environmentally friendly, efficient, and low-cost, providing a high-quality strain and technical support for the industrial production of bioethanol. Attached Figure Description

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

[0025] Figure 1 A schematic diagram of CRISPR / CAS9 gene editing;

[0026] Figure 2 The image shows the PCR detection results of strain M::X-3LysKB317::XII-1GA-CAS9; where M is the marker; 1 and 2 are strain M::X-3LysKB317-CAS9; 3 and 4 are strain M::X-3LysKB317::XII-1GA-CAS9; wt is strain M.

[0027] Figure 3 SDS-page analysis diagram of strain M::X-3LysKB317::XII-1GA-CAS9; where 1 is the supernatant of fermentation broth of strain M::X-3LysKB317::XII-1GA-CAS9; M is the negative control;

[0028] Figure 4The figure shows the alcohol content detection results of the fermentation experiment of corn mash fermentation medium with and without exogenous saccharifying enzyme; where M- represents M bacteria under the condition of no exogenous saccharifying enzyme addition; M+ represents M bacteria under the condition of exogenous saccharifying enzyme addition; MGA-Lys-CAS9- represents M::X-3LysKB317::XII-1GA-CAS9 under the condition of no exogenous saccharifying enzyme addition; MGA-Lys-CAS9+ represents M::X-3LysKB317::XII-1GA-CAS9 under the condition of exogenous saccharifying enzyme addition;

[0029] Figure 5 The cumulative mass loss over time in fermentation experiments of corn mash fermentation media with and without exogenous saccharifying enzyme is shown in the figure. Where M- represents strain M without exogenous saccharifying enzyme; M+ represents strain M with exogenous saccharifying enzyme; MGA-Lys-CAS9- represents M::X-3LysKB317::XII-1GA-CAS9 without exogenous saccharifying enzyme; MGA-Lys-CAS9+ represents M::X-3LysKB317::XII-1GA-CAS9 with exogenous saccharifying enzyme.

[0030] Figure 6 A statistical chart showing the residual reducing sugar content in corn mash fermentation media with and without exogenous saccharifying enzymes. Wherein, M- represents strain M without exogenous saccharifying enzyme; M+ represents strain M with exogenous saccharifying enzyme; MGA-Lys-CAS9- represents M::X-3LysKB317::XII-1GA-CAS9 without exogenous saccharifying enzyme; MGA-Lys-CAS9+ represents M::X-3LysKB317::XII-1GA-CAS9 with exogenous saccharifying enzyme. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] This invention utilizes CRISPR / CAS9 gene editing technology (the principle is as follows). Figure 1 As shown in the figure, the peptidoglycan hydrolase gene LysKB317 from bacteriophage and the saccharifying enzyme gene GA from *Saccharomyces cerevisiae* were heterologously integrated and expressed in *Saccharomyces cerevisiae*. The growth performance of the transformant strain and the antibacterial, saccharifying, and tolerance properties of subsequent strains were verified, and finally, a bifunctional antibacterial and saccharifying *Saccharomyces cerevisiae* strain was successfully obtained.

[0037] The experimental materials involved in the following examples are as follows:

[0038] Saccharomyces cerevisiae AMCC 30450 (M strain) was deposited at the China Center for Type Culture Collection (CCTCC) on September 8, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251982.

[0039] Saccharomyces cerevisiae 1522 has been disclosed in patent document CN105985969A, entitled “Genetically engineered yeast with saccharification function and its preparation and application”. It was deposited at the China Center for Type Culture Collection on December 23, 2014, with accession number CCTCC M 2014657.

[0040] The pH-CAS9 plasmid and pSCM-N20 plasmid have been published in the literature “Xu N, Chen H, Zhang Y, Yang Y, Wang Y, Liao B, et al. Metabolic Engineering and Genome-Wide Adaptive Evolution for Efficient Reduction of Glycerol in Industrial Saccharomyces cerevisiae. Synthetic Biology and Engineering 2025, 3, 10004”.

[0041] The Pscm-XII-1N20-V3 plasmid was obtained by fusing the guideRNA XII-1 sequence (SEQ ID NO.1) to the MCS sequence of the pSCM-N20 plasmid.

[0042] SEQ ID NO. 1: GTGGAGCAAATAATGAGCACAGG.

[0043] The Pscm-X-3N20-V3 plasmid was obtained by fusing the guideRNA X-3 sequence (SEQ ID NO.2) to the MCS sequence of the pSCM-N20 plasmid.

[0044] SEQ ID NO. 2: CTAATGTGTCCGCGTTTCTAAGG.

[0045] YPD medium (10 g / L yeast extract, 20 g / L tryptone, 20 g / L glucose; solid medium with 20 g / L agar added) was used for yeast strain culture. Resistant strains were screened on YPD agar plates containing 50 μg / mL Knowles sulfate and 500 μg / mL hygromycin. LB medium (10 g / L sodium chloride, 10 g / L tryptone, 5 g / L yeast extract) was used for Escherichia coli culture, MRS medium was used for lactic acid bacteria culture, fermentation medium (100 g / L sucrose, 20 g / L yeast extract FM888, 1 g / L magnesium sulfate heptahydrate, 1 g / L potassium dihydrogen phosphate) was used for yeast fermentation culture, and corn mash medium was used for yeast strain fermentation performance verification. Saccharomyces cerevisiae strains were cultured on a shaker at 30℃ (200 rpm), and Escherichia coli were cultured on a shaker at 37℃ (200 rpm). Lactic acid bacteria were cultured statically at 37℃.

[0046] Example 1: Strain Construction

[0047] 1. Heterologous expression of LysKB317

[0048] The peptidoglycan hydrolase gene LysKB317 (SEQ ID NO.3) from bacteriophages was integrated into M bacteria using CRISPR / CAS9 gene editing technology. The CRISPR / CAS9 gRNA plasmid was the Pscm-X-3N20-V3 plasmid constructed in previous studies; the LysKB317 repair fragment (SEQ ID NO.4; including 40 bp upstream and 40 bp downstream of the insertion site region) was amplified.

[0049] The pH-CAS9 plasmid, Pscm-X-3N20-V3 plasmid, and LysKB317 repair fragment were transformed into M bacteria using the lithium acetate transformation method.

[0050] The yeast cell preparation steps are as follows: Streak the bacterial strain onto a YPD plate and activate it overnight at 30℃; inoculate an appropriate amount of cells into 5 mL of YPD liquid medium and pre-culture at 30℃ and 200 r / min for 24 h; transfer 1 mL of bacterial culture to 50 mL of YPD liquid medium and incubate at 30℃ and 200 r / min for 5-6 h; when OD... 600 Once the bacterial culture reaches 0.6-0.8, collect all bacterial culture and centrifuge at 8000×g for 2 min. Discard the supernatant, wash the cell pellet twice with sterile water and once with 0.1M lithium acetate, and resuspend in 300 μL of 0.1M lithium acetate.

[0051] The transformation system was as follows: In a 1.5 mL centrifuge tube, add 240 μL of 50% PEG3350, 36 μL of 1M lithium acetate, 50 μL of boiled salmon sperm DNA (2 mg / mL), 400 ng of pH-CAS9 plasmid, 600 ng of Pscm-X-3N20-V3 plasmid, 1300 ng of LysKB317 repair fragment, and finally add 100 μL of bacterial suspension. After gently mixing the transformation solution, incubate at 30℃ for 30 min, then heat shock at 42℃ for 40 min. Centrifuge the transformation solution at 8000×g for 2 min, discard the supernatant, wash the cell pellet twice with sterile water, resuspend in 1 mL of YPD medium, and incubate at 30℃ and 200 rpm for 2–4 h. Centrifuge the bacterial culture and discard the supernatant. Resuspend the cell pellet in 200 μL of sterile water. Spread all bacterial cultures on YPD+Nours+hyg plates and incubate at 30℃ for 2 days.

[0052] All transformants were selected and verified by colony PCR (using the primer pair X-3-WF / X-3-WR shown in Table 1), sequencing confirmation, and plasmid removal to obtain the LysKB317 heterologous expression strain M::X-3LysKB317-CAS9.

[0053] Table 1 Primer Sequences

[0054]

[0055] 2. Heterologous expression of GA

[0056] The exogenous glucoamylase gene GA (SEQ ID NO.5) was site-directedly integrated into M::X-3LysKB317-CAS9:

[0057] The gRNA plasmid for CRISPR / CAS9 was the Pscm-XII-1N20-V3 plasmid constructed in previous studies. Using the S. cerevisiae 1522 genome as a template, the GA repair fragment (SEQ ID NO.6; including 40 bp upstream and 40 bp downstream of the insertion site region) was amplified using primer pair XII-1-ADH1p-F / XII-1-PDCt-R.

[0058] The pH-CAS9 plasmid, pscm-XII-1N20-V3 plasmid, and GA repair fragment were transformed into M::X-3LysKB317-CAS9. The transformation method was the same as in "1. Heterologous Expression of LysKB317". The transformed strains were verified by colony PCR (using primer pairs XII-1-WF and XII-1-WR) to obtain the recombinant Saccharomyces cerevisiae strain M::X-3LysKB317::XII-1GA-CAS9. The PCR verification results of strain M::X-3LysKB317::XII-1GA-CAS9 are shown in [link to PCR results]. Figure 2 The results showed that both the glucoamylase and peptidoglycan hydrolase genes were successfully integrated into strain M.

[0059] Example 2: Strain Performance Test

[0060] 1. Experimental Methods

[0061] 1.1 Starch hydrolysis and SDS-PAGE analysis

[0062] The recombinant Saccharomyces cerevisiae strain was inoculated into 3 mL of YPD medium and cultured overnight at 30°C and 200 rpm until OD500 was reached. 600 The concentration reached 4.7. Subsequently, 10 μL of the culture supernatant was inoculated onto an amylase activity plate and incubated at 30°C for 72 h. After incubation, approximately 2 mL of Gram's iodine solution (0.1% I₂, 1% KI) was poured into the starch degradation test plate and incubated for 15 minutes. Excess iodine solution was then discarded at room temperature. A clear hydrolytic zone formed around the recombinant strain, indicating the presence of amylase activity.

[0063] The recombinant Saccharomyces cerevisiae strain was inoculated into 3 mL of YPD medium and cultured overnight at 30°C and 200 rpm. After activation, the strain was inoculated into a shake flask containing 50 mL of YPD liquid medium at a 2% inoculation rate and cultured at 30°C and 180 rpm for 2 days with shaking. Subsequently, all bacterial culture was collected and centrifuged (6000 rpm, 5 min). 40 mL of the supernatant was then filtered through a 10 kDa ultrafiltration tube (6000 rpm, 10 min, 4°C). The sample in the collection tube was added to SDS-PAGE loading buffer, and the protein was separated on a 12% SDS-PAGE gel.

[0064] 1.2 Genetic stability and enzyme activity analysis

[0065] 200 μL of frozen M, M::X-3LysKB317::XII-1GA-CAS9 glycerol bacteria were inoculated into 5 mL of YPD medium and cultured overnight at 30℃ and 200 r / min. Then, the culture was streaked on YPD plates, and single colonies were picked and streaked on new YPD plates for subculturing. This process was repeated 30 times. Colonies from the 10th, 20th and 30th generations were selected, and their starch hydrolysis activity was determined by iodine staining.

[0066] To quantitatively analyze saccharifying enzymes, the strain was inoculated into YPD tubes and cultured overnight at 30°C and 200 r / min. 400 μL of the bacterial culture was then inoculated into 100 mL of YPD medium and cultured at 30°C and 200 r / min for 72 h. The fermentation broth was centrifuged at 5000 r / min for 5 min, and the resulting supernatant was used for saccharifying enzyme activity analysis. The reaction system consisted of 200 μL supernatant, 400 μL 2% starch solution, and 400 μL acetate-sodium acetate buffer (pH=4.6). The mixture was incubated at 40°C for 10 min, then heated in boiling water for 10 min to terminate the reaction. A control sample was incubated in boiling water for 10 min to inactivate the enzyme, and then reacted under the same conditions. The reducing sugar content was determined using the dinitrosalicylic acid method. One unit of enzyme activity is defined as the amount of enzyme that produces 1 μM reducing sugar (equivalent to 1 μM glucose) per minute under standard assay conditions.

[0067] 1.3 Application of microbial strains

[0068] 1.3.1 Validation of antibacterial ability

[0069] Three strains of lactic acid bacteria, activated by streaking, were cultured overnight in MRS liquid tubes; the OD of the bacterial culture was then measured. 600 Adjust the temperature to 0.6 for later use. Inoculate the control strain (wild-type M) and the experimental strain (M::X-3LysKB317::XII-1GA-CAS9) into the fermentation medium and incubate for 48 h.

[0070] The inhibition zone experiment was conducted using the Oxford cup method: (1) Pour an appropriate amount of YPD base medium into the plate; (2) After the base medium solidifies, place it in an Oxford cup and aspirate the OD. 600 Add the lactic acid bacteria adjusted to 0.6-0.8 to the corresponding dissolved and cooled upper culture medium at room temperature (addition amount is 1 / 10), shake evenly and then pour into the base culture medium containing Oxford cups until cooled and solidified, then remove the Oxford cups; (3) Centrifuge the fermentation broth of the control strain or experimental strain and inject 50 μL of the supernatant into the well, and culture under the corresponding culture conditions.

[0071] 1.3.2 Verification of saccharification ability

[0072] The bacterial strain was inoculated into YPD test tubes and cultured overnight at 30°C and 200 r / min. All bacterial cultures were then transferred to 600 mL of seed culture medium and cultured overnight at 30°C and 200 r / min. All bacterial cultures were then centrifuged at 5000 r / min for 5 min, and the supernatant was discarded. An equal mass of sterile water was added and mixed thoroughly by pipetting.

[0073] Corn mash fermentation medium was used, but no exogenous saccharifying enzymes were added. Inoculation was performed using fresh yeast at 25% dry weight. The mixture was cultured at 33°C and 170 rpm on a shaker. Weight loss was measured daily, and alcohol content was determined at the end of fermentation.

[0074] 1.3.3 Exogenous addition of lactic acid bacteria for fermentation

[0075] The method is the same as in 1.3.2, except that the corn mash culture medium needs to be supplemented with exogenous saccharifying enzymes. The experimental group was supplemented with 30 million / mL of Lactobacillus hilgardii, while the control group was not supplemented with lactic acid bacteria.

[0076] 2. Results and Analysis

[0077] 2.1 SDS-page analysis results

[0078] This invention performed SDS-page analysis on the fermentation broth supernatant of strain M::X-3LysKB317::XII-1GA-CAS9 ( Figure 3 The presence of protein bands at 57.4 kDa and 33 kDa indicates that both glucoamylase and peptidoglycan cleavage protein were successfully expressed and secreted normally into the extracellular space.

[0079] 2.2 Antibacterial performance test

[0080] This invention conducted antibacterial tests on strain M::X-3LysKB317::XII-1GA-CAS9. As shown in Table 2, the bifunctional yeast has a good antibacterial effect on lactic acid bacteria.

[0081] Table 2. Statistics on the diameter of the inhibition zone

[0082]

[0083] 2.3 Results of glycosylation level detection

[0084] This invention conducted fermentation experiments on corn mash fermentation media with and without exogenous saccharifying enzymes, and the results are as follows: Figure 4 As shown, without the addition of exogenous saccharifying enzyme, the final alcohol content of strain M::X-3LysKB317::XII-1GA-CAS9 was 15.7%, while the final fermentation alcohol content of the wild-type strain M, which does not contain the exogenous saccharifying enzyme gene, was only 1.4%. That is, after integrating the exogenous saccharifying enzyme gene, without the addition of exogenous saccharifying enzyme, the fermentation alcohol content of the transformant strain M::X-3LysKB317::XII-1GA-CAS9 was increased by about 200% compared with the original wild-type strain.

[0085] like Figure 5 As shown, the fermentation mass loss curves were analyzed. The cumulative mass loss of strain M::X-3LysKB317::XII-1GA-CAS9 was 36.59 g, while the cumulative mass loss of wild-type strain M was 8.32 g.

[0086] like Figure 6 As shown, the residual sugar content in the fermentation broth of wild-type strain M after 72 h of fermentation was 438.3 g / mL, while the residual sugar content in the fermentation broth of strain M::X-3LysKB317::XII-1GA-CAS9 after 72 h of fermentation was 60.92 g / mL, indicating that the saccharifying yeast significantly improved the utilization rate of residual sugar in the fermentation broth.

[0087] In summary, strain M::X-3LysKB317::XII-1GA-CAS9 exhibited good saccharification and antibacterial properties.

[0088] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for constructing a bifunctional antibacterial and saccharifying strain M::X-3LysKB317::XII-1GA-CAS9, characterized in that, The process includes the step of integrating the LysKB317 gene and the GA gene into the genome of the original Saccharomyces cerevisiae using the CRISPR / CAS9 gene editing method to construct the antibacterial saccharification bifunctional strain M::X-3LysKB317::XII-1GA-CAS9. The nucleotide sequence of the LysKB317 gene is shown in SEQ ID NO.3; the nucleotide sequence of the GA gene is shown in SEQ ID NO.

5.

2. The construction method according to claim 1, characterized in that, The initial brewing yeast was brewing yeast AMCC30450, whose accession number at the China Center for Type Culture Collection (CCTCC) was M 20251982.

3. The construction method according to claim 2, characterized in that, The construction method includes the following steps: The LysKB317 gene was integrated into the genome of the original Saccharomyces cerevisiae using the CRISPR / CAS9 gene editing method to obtain the recombinant strain M::X-3LysKB317-CAS9; The GA gene was integrated into the genome of the recombinant strain M::X-3LysKB317-CAS9 using the CRISPR / CAS9 gene editing method, resulting in the antibacterial glycosylation bifunctional strain M::X-3LysKB317::XII-1GA-CAS9.

4. The construction method according to claim 3, characterized in that, The method of integrating the LysKB317 gene into the genome of the initial Saccharomyces cerevisiae includes the steps of transforming the pH-CAS9 plasmid, the Pscm-X-3N20-V3 plasmid and the LysKB317 repair fragment into the initial Saccharomyces cerevisiae; The nucleotide sequence of the LysKB317 repair fragment is shown in SEQ ID NO.

4.

5. The construction method according to claim 4, characterized in that, The Pscm-X-3N20-V3 plasmid was obtained by fusing the guideRNA X-3 sequence to the MCS sequence of the pSCM-N20 plasmid; the nucleotide sequence of the guideRNA X-3 sequence is shown in SEQ ID NO.

2.

6. The construction method according to claim 3, characterized in that, The method of integrating the GA gene into the genome of the recombinant strain M::X-3LysKB317-CAS9 includes the steps of transforming the pH-CAS9 plasmid, pscm-XII-1N20-V3 plasmid and GA repair fragment into the recombinant strain M::X-3LysKB317-CAS9; The nucleotide sequence of the GA repair fragment is shown in SEQ ID NO.

6.

7. The construction method according to claim 6, characterized in that, The Pscm-XII-1N20-V3 plasmid was obtained by fusing the guideRNA XII-1 sequence to the MCS sequence of the pSCM-N20 plasmid; the nucleotide sequence of the guideRNA XII-1 sequence is shown in SEQ ID NO.

1.

8. A bifunctional antibacterial saccharified strain M::X-3LysKB317::XII-1GA-CAS9 constructed by the construction method according to any one of claims 1-7.

9. The application of the antibacterial and saccharifying bifunctional strain M::X-3LysKB317::XII-1GA-CAS9 as described in claim 8 in the fermentation production of ethanol.

10. A method for producing ethanol by fermentation, characterized in that, The step includes fermentation to produce ethanol using the antibacterial and saccharifying bifunctional strain M::X-3LysKB317::XII-1GA-CAS9 as described in claim 8.

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