High-temperature-resistant saccharomyces cerevisiae strain as well as construction method and application thereof

Abstract

The invention relates to a high-temperature-resistant saccharomyces cerevisiae strain and a construction method and application thereof in the field of microorganisms. The high-temperature-resistant saccharomyces cerevisiae strain disclosed by the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.27605, and the preservation name is SEB22. The SEB22 bacterial strain is obtained by knocking out an L-asparaginase II coding gene ASP3 and a high-expression transcription factor CRZ1 from an original bacterial strain SEB4. The glucose consumption rate, the ethanol yield and the yield of the SEB22 strain under high-temperature stress are obviously improved, and the SEB22 strain has good application potential in the aspect of high-temperature SSF fermentation.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and more particularly to a high temperature resistant brewer's yeast strain and a construction method and application thereof. Background Art

[0002] As energy crisis and environmental pollution become increasingly serious, renewable clean energy fuel ethanol has received great attention. Compared with food raw materials such as corn and cassava, the production of fuel ethanol using cellulosic raw materials such as straw is the main development direction of fuel ethanol in my country. Straw is converted into ethanol through pretreatment, enzymatic hydrolysis, fermentation and other steps. In order to improve production efficiency, simultaneous saccharification and fermentation (SSF) is usually used for production. However, there are significant differences between the optimal temperature for enzymatic hydrolysis (45℃~50℃) and the optimal temperature for fermentation (30℃~35℃), resulting in high cooling costs and low fermentation efficiency. Increasing the SSF temperature is an effective way to reduce production costs and improve fermentation efficiency. However, yeast cells face high temperature stress during high-temperature SSF, which seriously hinders cell growth and fermentation. Therefore, breeding Saccharomyces cerevisiae strains with excellent high temperature tolerance will help increase ethanol production and reduce production costs. Summary of the invention

[0003] In order to solve the above problems existing in the prior art, the present invention provides a thermostable brewer's yeast strain, and specifically relates to a thermostable brewer's yeast strain and a construction method and application thereof.

[0004] During the breeding process of high temperature resistant strains, the inventor team found that the transcription factor CRZ1 and the L-asparaginase II encoding gene ASP3 play an important role in affecting the high temperature tolerance of Saccharomyces cerevisiae. After a lot of practice, the inventor found that if SEB4 is used as the starting strain, CRZ1 and ASP3 are jointly regulated at the same time, which will have obvious advantages in improving the high temperature tolerance of the strain. Therefore, this application uses SEB4 (CGMCC11324) as the starting strain, and uses CRISPR / Cas9 gene editing technology to collaboratively knock out ASP3 and overexpress CRZ1 to obtain the engineered strain SEB22 (Saccharomyces cerevisiaeSEB22). Compared with the starting strain, the glucose consumption rate, ethanol production and yield of this strain under high temperature stress are significantly improved.

[0005] One of the purposes of the present application is to provide a thermostable Saccharomyces cerevisiae strain, which is deposited in the General Microbiological Center of China Microbiological Culture Management Committee with a deposit number of CGMCC NO.27605 and a deposit name of SEB22.

[0006] The second purpose of this application is to provide a method for constructing the thermostable Saccharomyces cerevisiae strain. The construction method is to use SEB4 as the starting strain, and use a gene editing method to knock out the L-asparaginase II encoding gene ASP3 and the highly expressed transcription factor CRZ1 to obtain the engineered strain SEB22; wherein, the starting strain SEB4 is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, and the deposit number is CGMCC NO.11324. wherein, the gene editing method can be a CRISPR / Cas9 gene editing method.

[0007] Specifically,

[0008] The method for constructing the thermostable Saccharomyces cerevisiae strain may include the following steps:

[0009] 1) Construction of gRNA plasmids pMEL13-ASP3 and pMEL13-CRZ1;

[0010] 2) construct repair fragments for ASP3 knockout and CRZ1 overexpression;

[0011] 3) Preparation of Cas9-NAT plasmid;

[0012] 4) Perform CRISPR / Cas9 transformation on the starting strain.

[0013] Wherein, the CRISPR / Cas9 transformation may include the following steps:

[0014] 1) introducing the Cas9-NAT plasmid into the starting strain to obtain a yeast strain containing the Cas9-NAT plasmid; the introduction method is preferably a lithium acetate transformation method;

[0015] 2) Knockout of the coding gene ASP3;

[0016] 3) High expression of transcription factor CRZ1.

[0017] Wherein, the method for knocking out the coding gene ASP3 may include the following steps:

[0018] The pMEL13-ASP3 plasmid and the repair fragment for ASP3 knockout were transferred into the yeast strain already containing the Cas9-NAT plasmid, and the plasmid was removed from the transformant to obtain the SEB4 ASP3 knockout strain.

[0019] The method for highly expressing the transcription factor CRZ1 may include the following steps:

[0020] The endogenous promoter of CRZ1 was replaced with the TEF1 promoter to highly express the transcription factor CRZ1;

[0021] Preferably, the method may specifically include the following steps: first, introducing the Cas9-NAT plasmid into the ASP3 knockout strain of SEB4; further introducing the pMEL13-CRZ1 plasmid and a repair fragment for high expression of CRZ1 into the ASP3 knockout strain of SEB4 containing the Cas9-NAT plasmid, and performing plasmid removal on the transformant to obtain the ASP3 knockout and CRZ1 high expression strain SEB22.

[0022] The third object of the present application is to provide a bacterial agent prepared by the high-temperature resistant brewing yeast strain described in the first object of the present application or the high-temperature resistant brewing yeast strain prepared according to the construction method described in the second object of the present application.

[0023] A fourth object of the present application is to provide an application of the thermostable Saccharomyces cerevisiae strain or the thermostable Saccharomyces cerevisiae strain prepared according to the construction method, preferably an application in material fermentation under stress conditions.

[0024] Wherein, the stress condition includes high temperature stress.

[0025] Strain deposit information

[0026] The thermostable Saccharomyces cerevisiae strain is deposited in the General Microbiology Center of China Microbiological Culture Management Committee, with the deposit number: CGMCC NO.27605, the deposit name: SEB22, the classification name: Saccharomyces cerevisiae, and the deposit date: June 12, 2023. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Evaluation of 44°C high temperature fermentation of different strains in Example 2 (vertical axis: cell dry weight, horizontal axis: fermentation time)

[0028] Figure 2 Evaluation of 44°C high temperature fermentation of different strains in Example 2 (the ordinate is: glucose concentration, ethanol concentration, the abscissa is: fermentation time) DETAILED DESCRIPTION

[0029] The present invention is described in detail below in conjunction with specific embodiments. It is necessary to point out that the following embodiments are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made to the present invention by those skilled in the art based on the contents of the present invention still fall within the scope of protection of the present invention.

[0030] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0031] Source of raw materials

[0032] The raw materials used in the examples and comparative examples, unless otherwise specified, are disclosed in the prior art, for example, they can be directly purchased or prepared according to the preparation methods disclosed in the prior art.

[0033] Example 1 Construction method

[0034] 1. Starting strain

[0035] The starting strain SEB4 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No.11324.

[0036] 2. Culture medium

[0037] The culture medium used is shown in Table 1. If it is a solid culture medium, add 2% agar powder before sterilization. The sterilization conditions are 0.1MPa, 121℃, 15min. After the culture medium is cooled to 50-60℃, add antibiotics.

[0038] Table 1 Culture medium composition

[0039]

[0040] 3. Plasmids, strains and primers

[0041] The plasmids and strains used are shown in Table 2; the primers used to construct the gRNA plasmids are shown in Table 3; the primers and fragments used for strain transformation are shown in Table 4.

[0042] Table 2 Plasmid and strain information

[0043]

[0044]

[0045] Table 3 Primers for constructing gRNA plasmids

[0046]

[0047] Note: F: upstream primer; R: downstream primer; Underline: 20bp recognition sequence.

[0048] The sequence number of CRZ1 tgR F is SEQ ID No.1, the sequence number of CRZ1 tgR R is SEQ ID No.2, the sequence number of ASP3tgR F is SEQ ID No.3, the sequence number of ASP3 tgR R is SEQ ID No.4, the sequence number of 6006-F is SEQ ID No.5, and the sequence number of 6005-R is SEQ ID No.6.

[0049] Table 4 Primers and fragments required for strain transformation

[0050]

[0051]

[0052] Note: RE: repair fragment; dg: verification primer; F: upstream primer; R: downstream primer

[0053] The sequence number of CRZ1 RE F is SEQ ID No.7, the sequence number of CRZ1 RE R is SEQ ID No.8, the sequence number of CRZ1 dgF is SEQ ID No.9, the sequence number of CRZ1 dg R is SEQ ID No.10, the sequence number of ASP3 RE F is SEQ ID No.11, the sequence number of ASP3 RE R is SEQ ID No.12, the sequence number of ASP3 dg F is SEQ ID No.13, the sequence number of ASP3dg R is SEQ ID No.14, the sequence number of Cas9-dg-F is SEQ ID No.15, and the sequence number of Cas9-dg-R is SEQ ID No.16.

[0054] 4 Construction of strain SEB22

[0055] 4.1 Construction of gRNA plasmids pMEL13-ASP3 and pMEL13-CRZ1

[0056] 1) Construction of double-stranded gRNA fragments

[0057] Referring to the genome information of the model yeast S288c, the FASTA sequences of the ASP3 coding region and the CRZ1 promoter region were inputted at the website http: / / www.e-crisp.org / E-CRISP / to obtain the 20 bp gRNA recognition sequence. Genewise was commissioned to synthesize the primer pairs ASP3 tgR F / R and CRZ1tgR F / R (sequences in Table 3) with 20 bp recognition sequences and 50 bp homology arms upstream and downstream. The two single-stranded primers were diluted to 10 μM with sterile water, mixed in equal volumes, heated at 95°C for 5 min, and cooled at room temperature to obtain double-stranded gRNA fragments.

[0058] 2) Amplify the linear backbone of the gRNA plasmid

[0059] The linear backbone was amplified using the pMEL13 plasmid as a template, and the PCR reaction system and reaction conditions were shown in Table 5. The PCR product was purified using the PCR product purification kit of OMEGA to obtain the linear backbone of the gRNA plasmid.

[0060] Table 5 PCR amplification of gRNA linear backbone

[0061]

[0062] The template plasmid was digested with Quick cut Dpn I. The digestion system and reaction conditions are shown in Table 6. The amount of DpnI used was determined by the amount of template plasmid (1 μg plasmid can be added with 1 μL DpnI). The gRNA linear backbone was purified.

[0063] Table 6 gRNA linear backbone template digestion system

[0064]

[0065] 3) Gibson ligation of gRNA fragments and linear backbone

[0066] The gRNA fragment and the pMEL13 linear backbone were connected by homologous recombination using the Gibson ligation kit. The Gibson ligation reaction system and conditions are shown in Table 7. Take 5 μL of the ligation solution and transform it into Escherichia coli. The transformation solution was spread on the LB+Kan plate and cultured at 37°C overnight. The transformant was inoculated in a test tube containing 5 mL of LB+Kan liquid culture medium and cultured for 12 to 16 hours (160 rpm, 37°C), the bacteria were collected, and the gRNA plasmid was extracted using the plasmid extraction kit of Omega Company. The correct gRNA plasmids pMEL13-ASP3 and pMEL13-CRZ1 were confirmed by sequencing.

[0067] Table 7 Gibson ligation reaction system

[0068]

[0069] 4.2 Building repair fragments

[0070] 1) Repair fragment for ASP3 knockout

[0071] The repair fragment for ASP3 knockout consists of 60 bp upstream and 60 bp downstream of the ASP3 coding sequence, and was commissioned to be synthesized in the form of primers by Genewise (sequences are shown in Table 4). The two single-stranded primers were diluted to 10 μM with sterile water, mixed in equal volumes, heated at 95°C for 5 min, and cooled at room temperature to obtain the repair fragment for ASP3 knockout, which can be directly used for yeast transformation.

[0072] 2) Repair fragment for high expression of CRZ1

[0073] The endogenous promoter of CRZ1 was replaced with the TEF1 promoter to achieve high expression of CRZ1. Using the genomic DNA of strain SEB4 as a template, primers CRZ1 RE F and CRZ1 RE R were used to amplify the repair fragment containing the TEF1 promoter sequence and homology arms (primer sequence is shown in Table 4, TEF1 promoter sequence is shown in Table 8). The PCR reaction system and reaction conditions are shown in Table 9. The PCR product was purified and used for transformation.

[0074] Table 8 TEF1 promoter sequence

[0075]

[0076]

[0077] P TEF1 The sequence number is SEQ ID No.17.

[0078] Table 9 PCR amplification repair fragment

[0079]

[0080] 4.3 Cas9-NAT plasmid preparation

[0081] E. coli containing the Cas9-NAT plasmid was inoculated into 5 mL of LB+NAT liquid medium and cultured at 37°C and 160 rpm for 12 to 16 hours. The bacteria were collected by centrifugation, and the Cas9-NAT plasmid was extracted using a plasmid extraction kit from OMEGA.

[0082] 4.4 CRISPR / Cas9 transformation

[0083] 1) Introduction of Cas9-NAT plasmid into Saccharomyces cerevisiae strains by lithium acetate transformation

[0084] a. Streak strain SEB4 on a 2% YPD plate for activation, take an appropriate amount of cells and inoculate into 5 mL 2% YPD liquid medium, and culture at 30°C and 160 rpm for 16 h;

[0085] b. Take 2 mL of bacterial solution and inoculate it into 100 mL of 2% YPD culture medium. Cultivate at 30°C and 160 rpm for 2 to 3 hours. 600 When the concentration reached 0.2-0.3, all the bacteria were collected by centrifugation, the bacterial precipitate was washed twice with sterile water, resuspended in 300 μL sterile water, and placed on ice for later use;

[0086] c. Boil salmon sperm DNA (ssDNA) at 100°C for 5 min and place on ice for later use;

[0087] d. Add 60% PEG4000 (115 μL), 4 M lithium acetate solution (5 μL), ssDNA (10 μL), Cas9-NAT plasmid (100 ng) and yeast cells (50 μL) in a 1.5 mL centrifuge tube, mix well and heat shock at 42°C for 40 min;

[0088] e. Centrifuge at 8,000 × g for 1 min, discard the supernatant, wash the cells twice with sterile water, add 1 mL of 2% YPD culture medium, and culture at 30°C and 160 rpm for 2 to 3 h;

[0089] f. Centrifuge at 8,000×g for 1 min, discard the supernatant, and resuspend the cells in 1 mL of sterile water. Spread 100 μL of the bacterial solution on a 2% YPD+NAT plate and culture at 30°C for 2 to 3 days;

[0090] g. Streak the transformants onto 2% YPD+NAT plates and perform colony PCR verification after culturing for 24 h.

[0091] 2) Colony PCR verification of Cas9-NAT plasmid in yeast transformants

[0092] a. Add 95 μL 1% SDS, 5 μL 4M lithium acetate solution and appropriate amount of bacteria to a 1.5 mL centrifuge tube, vortex and incubate at 75°C for 10 min;

[0093] b. Add 300 μL of anhydrous ethanol to the centrifuge tube and vortex; centrifuge at 13000 rpm for 3 min at room temperature, discard the supernatant, and dry at 37°C with the lid open for 10 min;

[0094] c. Add 100 μL sterile water, vortex, centrifuge at 13000 rpm for 1 min at room temperature, and retain the supernatant;

[0095] d. Determine the concentration of the supernatant, adjust the OD to 10-20 ng / μL, take 1 μL as a template for PCR verification, and the PCR reaction system and reaction conditions are shown in Table 10;

[0096] e. 1.5% agarose gel electrophoresis (100 V, 30 min) was used to verify the target band.

[0097] Table 10 Colony PCR Verification System

[0098]

[0099] 3) ASP3 knockout

[0100] The pMEL13-ASP3 plasmid and the knockout repair fragment were transferred into the yeast strain containing the Cas9-NAT plasmid. The transformation process was the same as 1). Nourseothricin (NAT) was added to the 2% YPD medium to ensure the stable presence of the Cas9-NAT plasmid. The transformation system is shown in Table 11. The cells were resuspended with 200 μL of sterile water, and all the bacterial liquid was spread on a 2% YPD+NAT+G418 plate and cultured at 30°C for 2 to 3 days. The transformants were picked for colony PCR verification. The specific method was the same as 2). The target fragment was amplified using primers ASP3 dg F and ASP3 dgR (Table 4). The PCR reaction system and reaction conditions are shown in Table 10. The extension time and annealing temperature were adjusted according to the characteristics of the primers. The PCR product with the correct band was sequenced for confirmation. The plasmid of the correct transformant was further removed.

[0101] Table 11 Target gene transformation system

[0102]

[0103] 4) Removal of plasmid

[0104] Remove the Cas9-NAT plasmid and gRNA plasmid of the correct transformant. The specific steps are as follows:

[0105] a. Streak the correct transformants onto 2% YPD plates, pick a small amount of cells and inoculate into 10 mL 2% YPD medium, and culture at 30°C for 1 day.

[0106] b. Take 200 μL of bacterial solution and dilute 10 5 times, take 100 μL of bacterial solution and spread it on 2% YPD plate, and culture for 1-2 days;

[0107] c. Take a small amount of cells and resuspend them in 200 μL sterile water (pick 5 to 8 single bacteria for each transformant), take 2 μL of the bacterial suspension and spot them on 2% YPD, 2% YPD+G418 and 2% YPD+NAT plates, and culture them at 30°C for 1-2 days; the strain that can only grow on 2% YPD plates has successfully removed the plasmid, and the ASP3 knockout strain of SEB4 (SEB19) is obtained.

[0108] 5) CRZ1 high expression

[0109] CRZ1 was highly expressed in SEB4 and its ASP3 knockout strain, respectively. First, Cas9-NAT plasmid was introduced into SEB4 and its ASP3 knockout strain, the method was the same as 1). Further, pMEL13-CRZ1 plasmid and repair fragment for high expression were introduced into SEB4 and its ASP3 knockout strain containing Cas9-NAT plasmid, the method was the same as 3). Colony PCR verification was performed using primers CRZ1 dg F and CRZ1 dg R (Table 4). The PCR reaction system and reaction conditions are shown in Table 10. The PCR products with correct bands were sequenced and confirmed. Plasmid removal was performed on the correct transformants, the method was the same as 4). Finally, the CRZ1 highly expressed strain SEB4-CRZ1 and the ASP3 knockout and CRZ1 highly expressed strain SEB22 of the present invention were obtained.

[0110] Example 2 High temperature fermentation

[0111] With glucose as the sole carbon source, the fermentation performance of SEB4 (starting strain), SEB19 (SEB4, ASP3 knockout), SEB4-CRZ1 (SEB4, CRZ1 overexpression) and SEB22 (SEB4, ASP3 knockout and CRZ1 overexpression) at 44°C was compared by batch fermentation in Erlenmeyer flasks. Figures 1-2 As shown in the results, under the conditions of initial glucose concentration of 143.5 g / L and high temperature of 44 °C, the four strains rapidly utilized glucose and produced ethanol within the first 24 h of fermentation, and glucose consumption slowed down after 24 h. At 72 h of fermentation, SEB4 consumed 94.14 g / L glucose and produced 41.23 g / L ethanol, with a yield of 0.44 g / g consumed sugar. By knocking out ASP3 on the basis of SEB4, the glucose consumption and ethanol production of strain SEB19 increased by 2.91% and 5.95%, respectively; by overexpressing CRZ1 on the basis of SEB4, the glucose consumption and ethanol production of the strain decreased by 9.02% and 5.15%, respectively; by synergistically knocking out ASP3 and overexpressing CRZ1 on the basis of SEB4, the glucose consumption and ethanol production of the strain increased by 11.93% and 16.47%, respectively, and the ethanol yield reached 0.46 g / g consumed sugar (Table 12). In addition, within 72 hours of fermentation, the accumulated cell dry weight of SEB19, SEB4-CRZ1 and SEB22 was 15.86%, 15.66% and 33.33% higher than that of SEB4, respectively (Table 12). Under stress conditions, the growth and metabolic capacity of the strain are very important, and it can be seen that the growth and metabolic capacity of the strain described in the present invention are significantly improved. In summary, the coordinated knockout of ASP3 and high expression of CRZ1 can significantly improve the high temperature tolerance of the strain.

[0112] The maximum tolerant temperature of conventional thermostable Saccharomyces cerevisiae strains reported in the prior art is 42°C (Table 13). In comparison, the SEB22 strain obtained in the present invention can tolerate a higher temperature (44°C) and has a higher ethanol yield. Compared with the strain SEB19 in the previous patent CN113717874A of this team, the glucose consumption and ethanol production of SEB22 under high temperature conditions of 44°C are unexpectedly significantly improved (Table 13). It can be seen that the SEB22 strain obtained in the present invention has great application potential in high temperature SSF fermentation.

[0113] Table 12 Strain performance of high temperature fermentation for 72h

[0114]

[0115] Note: t-test was used to test the statistical significance between groups, and all strains were compared with SEB4. *p<0.05, **p<0.01, ***p<0.001. SEB19: SEB4 knocked out ASP3; SEB4-CRZ1: SEB4 overexpressed CRZ1; SEB22: SEB4 knocked out ASP3 and overexpressed CRZ1.

[0116] Table 13 Comparison between the present application and the prior art of high temperature fermentation strains

[0117]

[0118] [1]Mans R, van Rossum HM, Wijsman M, et al. CRISPR / Cas9: a molecular swissarmy knife f or simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae[J]. FEMS Yeast Research, 2015, 15(2):fov004.

[0119] [2]Zhang GC, Kong II, Kim H, et al. Construction of a quadrupleauxotrophic mutant of an industrial polyploid Saccharomyces cerevisiae strain by using RNA-guided Cas9 nuclease[J]. Applied and Environmental Microbiology, 2014, 80(24):7694-7701.

[0120] [3]Auesukaree C, Koedrith P, Saenpayavai P, et al.Characterization and gene expression profiles of thermotolerant Saccharomyces cerevisiae isolates from Thai fruits[J].Journal of Bioscience and Bioengineering, 2012,114(2):144-149.

[0121] [4]Lu Y, Cheng YF, He XP, et al. Improvement of robustness and ethanolproduction of ethanologenic Saccharomyces cerevisiae under co-stress of heat and inhibitors[J]. Journal of Industrial Microbiology and Biotechnology, 2012, 39(1):73-80.

[0122] [5] Wu Youduo, Yuan Runpeng, Xue Chuang, Xu Yaqing, Song Yongxiu, Wang Huan. A high temperature, high sugar and high osmotic tolerance yeast and its breeding method and application in high concentration ethanol fermentation, CN115948263A[P].2022.

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[0124] [7] Tang Yueqin, Wang Li, Xie Caiyun, Gou Min, Sun Zhaoyong, Xia Ziyuan. A high temperature and high sugar resistant Saccharomyces cerevisiae strain and its construction method and application, CN113717874A[P].2021.

Claims

1. A thermostable Saccharomyces cerevisiae strain, characterized in that: The thermostable Saccharomyces cerevisiae strain is deposited in the General Microbiological Center of China Microbiological Culture Management Committee with a deposit number of CGMCC NO.27605 and a deposit name of SEB22.

2. The method for constructing a thermostable Saccharomyces cerevisiae strain according to claim 1, characterized in that: The L-asparaginase II encoding gene ASP3 and the highly expressed transcription factor CRZ1 were knocked out from the starting strain to obtain the strain SEB22; the starting strain was a Saccharomyces cerevisiae strain.

3. The method for constructing a thermostable Saccharomyces cerevisiae strain according to claim 2, characterized in that: The starting strain is strain SEB4, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with a deposit number of CGMCC NO.11324.

4. The method for constructing a thermostable Saccharomyces cerevisiae strain according to claim 2, characterized in that The following steps are involved: 1) Construction of gRNA plasmids pMEL13-ASP3 and pMEL13-CRZ1; 2) construct repair fragments for ASP3 knockout and CRZ1 overexpression; 3) Preparation of Cas9-NAT plasmid; 4) Perform CRISPR / Cas9 transformation on the starting strain.

5. The method for constructing a thermostable Saccharomyces cerevisiae strain according to claim 4, characterized in that: The CRISPR / Cas9 transformation comprises the following steps: 1) introducing the Cas9-NAT plasmid into the starting strain to obtain a yeast strain containing the Cas9-NAT plasmid; the introduction method is preferably a lithium acetate transformation method; 2) Knockout of the coding gene ASP3; 3) High expression of transcription factor CRZ1.

6. The method for constructing a thermostable Saccharomyces cerevisiae strain according to claim 5, characterized in that: The method for knocking out the coding gene ASP3 comprises the following steps: The pMEL13-ASP3 plasmid and the repair fragment for ASP3 knockout were transferred into the yeast strain already containing the Cas9-NAT plasmid, and the plasmid was removed from the transformant to obtain the SEB4 ASP3 knockout strain.

7. The method for constructing a thermostable Saccharomyces cerevisiae strain according to claim 5, characterized in that: The method for highly expressing the transcription factor CRZ1 comprises the following steps: The endogenous promoter of CRZ1 was replaced with the TEF1 promoter to highly express the transcription factor CRZ1; Preferably, the method comprises the following steps: First, the Cas9-NAT plasmid was introduced into the ASP3 knockout strain of SEB4. Then, the pMEL13-CRZ1 plasmid and the repair fragment for CRZ1 overexpression were introduced into the ASP3 knockout strain of SEB4 containing the Cas9-NAT plasmid, and the plasmid was removed from the transformant to obtain the ASP3 knockout and CRZ1 overexpression strain SEB22.

8. A bacterial agent prepared using the thermostable Saccharomyces cerevisiae strain according to claim 1 or the thermostable Saccharomyces cerevisiae strain prepared according to the construction method of any one of claims 2 to 7.

9. Use of the thermotolerant Saccharomyces cerevisiae strain according to claim 1 or the thermotolerant Saccharomyces cerevisiae strain prepared according to the construction method of any one of claims 2 to 7, preferably in fermentation of materials under stress conditions.

10. The use according to claim 9, wherein the stress condition comprises high temperature stress.

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