Saccharomyces cerevisiae recombinant strain for improving vanillin tolerance and construction method thereof

Abstract

The invention relates to the technical field of synthetic biology and metabolic engineering. The invention provides a saccharomyces cerevisiae recombinant strain capable of improving vanillin tolerance and a construction method thereof, which are characterized in that related genes MSH1, MSH2, MSH3, MSH4, MSH5, MSH6, MLH1, MLH2, MLH3 or PMS1 of a strain DNA mismatch repair system are innovatively knocked out, so that the adaptive evolution ability of the strain is enhanced, and the limitation of traditional metabolic engineering modification is broken through; a stepped vanillin concentration domestication strategy is utilized, and a mutant strain with remarkably improved tolerance is obtained through screening. The limitation of vanillin toxicity on thallus growth and product synthesis is effectively relieved, and a certain foundation is laid for constructing an efficient vanillin biosynthesis system.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a recombinant strain of Saccharomyces cerevisiae that improves vanillin tolerance and its construction method. Background Technology

[0002] Vanillin, a high-value flavoring agent, has a global annual demand exceeding 29 million tons. Breakthroughs in its biosynthesis technology are of great significance for mitigating the environmental pollution caused by chemical synthesis methods (approximately 5 tons of wastewater are generated per ton of product) and replacing high-cost natural extraction processes (costing approximately $3,000-$4,000 per kilogram). Currently, the key technological bottleneck in the microbial synthesis of vanillin is the inhibition of product toxicity. When the vanillin concentration exceeds 500 mg / L, it severely hinders the growth of brewing yeast (Mo, Q. & Yuan, J. Minimal aromatic aldehyde reduction (MARE) yeast platform for engineering vanillin production. BiotechnolBiofuels Bioprod. 2024, 17, 4). Although previous studies have shown that the glycosylation of vanillin can be achieved by introducing the glycosyltransferase UGT72B1, catalyzing the conversion of vanillin into non-toxic glucvanillin, this strategy requires an additional separation step (Xin, X. et al. Redesigned Pathway for De Novo Synthesis of Vanillin and Co-conversion of Multiple Renewable Substrates in Saccharomyces cerevisiae. JacsAu. 2025, 5, 1133-114).

[0003] Based on the above, it is crucial to overcome the bottlenecks in microbial synthesis and achieve industrialized bio-manufacturing of vanillin by mitigating the limitations imposed by vanillin toxicity on cell growth and product synthesis. Summary of the Invention

[0004] The purpose of this invention is to provide a recombinant strain of Saccharomyces cerevisiae that improves vanillin tolerance and its construction method. This recombinant strain can produce high tolerance to vanillin and has high activity.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a recombinant Saccharomyces cerevisiae strain that improves vanillin tolerance, using strain RARE12 as the original strain and knocking out the mismatch repair gene of strain RARE12.MSH1 , MSH2 , MSH3 , MSH4 , MSH5 , MSH6 , MLH1 , MLH2 , MLH3 or PMS1 The engineered strains were then domesticated in a concentration gradient culture medium containing vanillin.

[0006] Preferably, the strain RARE12 is: using Saccharomyces cerevisiae BY4741 as the original strain, and sequentially knocking out the aldehyde accumulation gene of Saccharomyces cerevisiae BY4741. AAD3 , ARI1 , YPR1 , YDR541C , GCY1 , SFA1 , YDL124W , GRE3 , GRE2 , ADH6 , ADH7 , HFD1 The obtained strain.

[0007] Preferably, the concentration gradient of vanillin is 0.5-1.7 g / L.

[0008] The present invention also provides a method for constructing the recombinant Saccharomyces cerevisiae strain that improves vanillin tolerance, comprising the following steps: (1) Using p426-SNR52-gRNA as a template, the gene was obtained by PCR amplification. MSH1 , MSH2 , MSH3 , MSH4 , MSH5 , MSH6 , MLH1 , MLH2 , MLH3 or PMS1 The gRNA expression fragments were then ligated to the enzyme-digested vector plasmids to construct recombinant plasmids p426-gRNA(MSH1), p426-gRNA(MSH2), p426-gRNA(MSH3), p426-gRNA(MSH4), p426-gRNA(MSH5), p426-gRNA(MSH6), p426-gRNA(MLH1), p426-gRNA(MLH2), p426-gRNA(MLH3), or p426-gRNA(PMS1). (2) The recombinant plasmid described in step (1) and the knockout integration fragment of the corresponding gene obtained by PCR amplification were transformed into RARE12 competent cells to obtain the engineered strain RARE12.ΔMSH1 ), RARE12 ΔMSH2 ), RARE12 ΔMSH3 ), RARE12 ΔMSH4 ), RARE12 ΔMSH5 ), RARE12 ΔMSH6 ), RARE12 ΔMLH1 ), RARE12 ΔMLH2 ), RARE12 ΔMLH3 ) or RARE12 ( ΔPMS1 ); (3) The bacterial culture of the engineered strain obtained in step (2) is inoculated into a culture medium containing 0.5 g / L vanillin to obtain the first generation culture. Then, the previous generation culture is transferred sequentially according to the concentration gradient of 0.75-1.7 g / L vanillin to the ninth generation culture, which is obtained.

[0009] Preferably, step (1) is described MSH1 The PCR amplification primers for the gene are F_gRNA.MSH1 as shown in SEQ ID NO:13 and R_SUP4 as shown in SEQ ID NO:23; MSH2 The PCR amplification primers for the gene were F_gRNA.MSH2 as shown in SEQ ID NO:14 and R_SUP4 as shown in SEQ ID NO:23; MSH3 The PCR amplification primers for the gene were F_gRNA.MSH3 as shown in SEQ ID NO:15 and R_SUP4 as shown in SEQ ID NO:23; MSH4 The PCR amplification primers for the gene are F_gRNA.MSH4 as shown in SEQ ID NO:16 and R_SUP4 as shown in SEQ ID NO:23; MSH5 The PCR amplification primers for the gene were F_gRNA.MSH5 as shown in SEQ ID NO:17 and R_SUP4 as shown in SEQ ID NO:23; MSH6 The PCR amplification primers for the gene were F_gRNA.MSH6 as shown in SEQ ID NO:18 and R_SUP4 as shown in SEQ ID NO:23; MLH1 The PCR amplification primers for the gene are F_gRNA.MLH1 as shown in SEQ ID NO:19 and R_SUP4 as shown in SEQ ID NO:23; MLH2 The PCR amplification primers for the gene are F_gRNA.MLH2 as shown in SEQ ID NO:20 and R_SUP4 as shown in SEQ ID NO:23; MLH3The PCR amplification primers for the gene are F_gRNA.MLH3 as shown in SEQ ID NO:21 and R_SUP4 as shown in SEQ ID NO:23; PMS1 The PCR amplification primers for the gene were F_gRNA.PMS1 as shown in SEQ ID NO:22 and R_SUP4 as shown in SEQ ID NO:23.

[0010] As a preferred option, step (2) gene MSH1 The PCR amplification primers for knocking out the integrated fragment are F-MSH1-Del as shown in SEQ ID NO:48 and R-MSH1-Del as shown in SEQ ID NO:49; gene MSH2 The PCR amplification primers for knocking out the integrated fragment are F-MSH2-Del as shown in SEQ ID NO:50 and R-MSH2-Del as shown in SEQ ID NO:51; gene MSH3 The PCR amplification primers for knocking out the integrated fragment are F-MSH3-Del as shown in SEQ ID NO:52 and R-MSH3-Del as shown in SEQ ID NO:53; gene MSH4 The PCR amplification primers for knocking out the integrated fragment are F-MSH4-Del as shown in SEQ ID NO:54 and R-MSH4-Del as shown in SEQ ID NO:55; gene MSH5 The PCR amplification primers for knocking out the integrated fragment are F-MSH5-Del as shown in SEQ ID NO:56 and R-MSH5-Del as shown in SEQ ID NO:57; gene MSH6 The PCR amplification primers for knocking out the integrated fragment are F-MSH6-Del as shown in SEQ ID NO:58 and R-MSH6-Del as shown in SEQ ID NO:59; gene MLH1 The PCR amplification primers for knocking out the integrated fragment are F-MLH1-Del as shown in SEQ ID NO:60 and R-MLH1-Del as shown in SEQ ID NO:61; gene MLH2 The PCR amplification primers for knocking out the integrated fragment are F-MLH2-Del as shown in SEQ ID NO:62 and R-MLH2-Del as shown in SEQ ID NO:63; gene MLH3 The PCR amplification primers for knocking out the integrated fragment are F-MLH3-Del as shown in SEQ ID NO:64 and R-MLH3-Del as shown in SEQ ID NO:65; gene PMS1The PCR amplification primers for knocking out the integrated fragment are F-PMS1-Del as shown in SEQ ID NO:66 and R-PMS1-Del as shown in SEQ ID NO:67.

[0011] Preferably, the culture medium in step (3) consists of: 6.5-7 g / L yeast nitrogen source base, 1.2-1.6 g / L yeast auxotrophic culture medium supplement and 18-22 g / L glucose.

[0012] Preferably, the amount of inoculation in step (3) is 5-10% v / v.

[0013] Preferably, the culture temperature in step (3) is 28-32℃.

[0014] This invention also provides genes related to strain mismatch repair systems. MSH1 , MSH2 , MSH3 , MSH4 , MSH5 , MSH6 , MLH1 , MLH2 , MLH3 or PMS1 Application in improving vanillin tolerance in brewer's yeast.

[0015] By adopting the above technical solution, the present invention has the following beneficial effects: The technical solution of this invention innovatively knocks out the DNA mismatch repair system (MMR) related genes in the RARE12 strain. MSH1 , MSH2 , MSH3 , MSH4 , MSH5 , MSH6 , MLH1 , MLH2 , MLH3 or PMS1 A single-gene deletion mutant library was constructed; and a stepwise concentration acclimatization strategy (vanillin gradient of 0.5-1.7 g / L) was used to screen for mutant strains with significantly improved tolerance. This technical approach not only enhances the adaptive evolutionary ability of the strains but also overcomes the limitations of traditional metabolic engineering. Experimental results show that the modified strain *Saccharomyces cerevisiae* RARE12 (… ΔMLH1 OD under 1.3 g / L vanillin stress 600 The maximum tolerance reached 1.0 g / L, while the control group could not grow under these conditions. After further acclimatization, the strain could tolerate up to 1.7 g / L vanillin. This invention effectively alleviates the limitations imposed by vanillin toxicity on bacterial growth and product synthesis, laying a foundation for constructing an efficient vanillin biosynthesis system. Attached Figure Description

[0016] Figure 1 This is a schematic diagram illustrating the construction principle of the recombinant Saccharomyces cerevisiae strain that enhances vanillin tolerance according to the present invention. Figure 2 Growth curves of different recombinant strains of *Saccharomyces cerevisiae* and control strains in SC-LEU medium containing 0.5 g / L, 0.75 g / L, 1.3 g / L, and 1.7 g / L vanillin, respectively. Figure 2 In the diagram, A represents a culture medium containing 0.5 g / L vanillin, B represents a culture medium containing 0.75 g / L vanillin, C represents a culture medium containing 1.3 g / L vanillin, and D represents a culture medium containing 1.7 g / L vanillin. Detailed Implementation

[0017] This invention provides a recombinant Saccharomyces cerevisiae strain that improves vanillin tolerance, using strain RARE12 as the original strain and knocking out the mismatch repair gene of strain RARE12. MSH1 , MSH2 , MSH3 , MSH4 , MSH5 , MSH6 , MLH1 , MLH2 , MLH3 or PMS1 The engineered strains were then domesticated in a concentration gradient culture medium containing vanillin.

[0018] In this invention, the strain RARE12 is: using Saccharomyces cerevisiae BY4741 as the original strain, the aldehyde accumulation gene of Saccharomyces cerevisiae BY4741 is knocked out sequentially. AAD3 , ARI1 , YPR1 , YDR541C , GCY1 , SFA1 , YDL124W , GRE3 , GRE2 , ADH6 , ADH7 , HFD1 The obtained strain.

[0019] In this invention, the concentration gradient of vanillin is preferably 0.5-1.7 g / L.

[0020] The present invention also provides a method for constructing the recombinant Saccharomyces cerevisiae strain that improves vanillin tolerance, comprising the following steps: (1) Using p426-SNR52-gRNA as a template, the gene was obtained by PCR amplification. MSH1 , MSH2 , MSH3 ,MSH4 , MSH5 , MSH6 , MLH1 , MLH2 , MLH3 or PMS1 The gRNA expression fragments were then ligated to the enzyme-digested vector plasmids to construct recombinant plasmids p426-gRNA(MSH1), p426-gRNA(MSH2), p426-gRNA(MSH3), p426-gRNA(MSH4), p426-gRNA(MSH5), p426-gRNA(MSH6), p426-gRNA(MLH1), p426-gRNA(MLH2), p426-gRNA(MLH3), or p426-gRNA(PMS1). (2) The recombinant plasmid described in step (1) and the knockout integration fragment of the corresponding gene obtained by PCR amplification were transformed into RARE12 competent cells to obtain the engineered strain RARE12. ΔMSH1 ), RARE12 ΔMSH2 ), RARE12 ΔMSH3 ), RARE12 ΔMSH4 ), RARE12 ΔMSH5 ), RARE12 ΔMSH6 ), RARE12 ΔMLH1 ), RARE12 ΔMLH2 ), RARE12 ΔMLH3 ) or RARE12 ( ΔPMS1 ); (3) The bacterial culture of the engineered strain obtained in step (2) is inoculated into a culture medium containing 0.5 g / L vanillin to obtain the first generation culture. Then, the previous generation culture is transferred sequentially according to the concentration gradient of 0.75-1.7 g / L vanillin to the ninth generation culture, which is obtained.

[0021] In this invention, step (1) is described MSH1 The PCR amplification primers for the gene are F_gRNA.MSH1 as shown in SEQ ID NO:13 and R_SUP4 as shown in SEQ ID NO:23; MSH2 The PCR amplification primers for the gene were F_gRNA.MSH2 as shown in SEQ ID NO:14 and R_SUP4 as shown in SEQ ID NO:23; MSH3 The PCR amplification primers for the gene were F_gRNA.MSH3 as shown in SEQ ID NO:15 and R_SUP4 as shown in SEQ ID NO:23; MSH4The PCR amplification primers for the gene are F_gRNA.MSH4 as shown in SEQ ID NO:16 and R_SUP4 as shown in SEQ ID NO:23; MSH5 The PCR amplification primers for the gene were F_gRNA.MSH5 as shown in SEQ ID NO:17 and R_SUP4 as shown in SEQ ID NO:23; MSH6 The PCR amplification primers for the gene were F_gRNA.MSH6 as shown in SEQ ID NO:18 and R_SUP4 as shown in SEQ ID NO:23; MLH1 The PCR amplification primers for the gene are F_gRNA.MLH1 as shown in SEQ ID NO:19 and R_SUP4 as shown in SEQ ID NO:23; MLH2 The PCR amplification primers for the gene are F_gRNA.MLH2 as shown in SEQ ID NO:20 and R_SUP4 as shown in SEQ ID NO:23; MLH3 The PCR amplification primers for the gene are F_gRNA.MLH3 as shown in SEQ ID NO:21 and R_SUP4 as shown in SEQ ID NO:23; PMS1 The PCR amplification primers for the gene were F_gRNA.PMS1 as shown in SEQ ID NO:22 and R_SUP4 as shown in SEQ ID NO:23.

[0022] In this invention, step (2) gene MSH1 The PCR amplification primers for knocking out the integrated fragment are F-MSH1-Del as shown in SEQ ID NO:48 and R-MSH1-Del as shown in SEQ ID NO:49; gene [[ID=1三十九]]MSH2 The PCR amplification primers for knocking out the integrated fragment are F-MSH2-Del as shown in SEQ ID NO:50 and R-MSH2-Del as shown in SEQ ID NO:51; gene MSH3 The PCR amplification primers for knocking out the integrated fragment are F-MSH3-Del as shown in SEQ ID NO:52 and R-MSH3-Del as shown in SEQ ID NO:53; gene MSH4 The PCR amplification primers for knocking out the integrated fragment are F-MSH4-Del as shown in SEQ ID NO:54 and R-MSH4-Del as shown in SEQ ID NO:55; gene MSH5 The PCR amplification primers for knocking out the integrated fragment are F-MSH5-Del as shown in SEQ ID NO:56 and R-MSH5-Del as shown in SEQ ID NO:57; gene MSH6The PCR amplification primers for knocking out the integrated fragment are F-MSH6-Del as shown in SEQ ID NO:58 and R-MSH6-Del as shown in SEQ ID NO:59; gene MLH1 The PCR amplification primers for knocking out the integrated fragment are F-MLH1-Del as shown in SEQ ID NO:60 and R-MLH1-Del as shown in SEQ ID NO:61; gene MLH2 The PCR amplification primers for knocking out the integrated fragment are F-MLH2-Del as shown in SEQ ID NO:62 and R-MLH2-Del as shown in SEQ ID NO:63; gene MLH3 The PCR amplification primers for knocking out the integrated fragment are F-MLH3-Del as shown in SEQ ID NO:64 and R-MLH3-Del as shown in SEQ ID NO:65; gene PMS1 The PCR amplification primers for knocking out the integrated fragment are F-PMS1-Del as shown in SEQ ID NO:66 and R-PMS1-Del as shown in SEQ ID NO:67.

[0023] In this invention, the culture medium in step (3) preferably consists of 6.5-7 g / L yeast nitrogen source basal, 1.2-1.6 g / L yeast auxotrophic culture medium supplement and 18-22 g / L glucose; more preferably 6.6-6.8 g / L yeast nitrogen source basal, 1.3-1.5 g / L yeast auxotrophic culture medium supplement and 19-21 g / L glucose; and even more preferably 6.7 g / L yeast nitrogen source basal, 1.4 g / L yeast auxotrophic culture medium supplement and 20 g / L glucose.

[0024] In this invention, the amount of inoculation in step (3) is preferably 5-10% v / v, more preferably 8% v / v, and even more preferably 10% v / v.

[0025] In this invention, the culture temperature in step (3) is preferably 28-32℃, more preferably 29-31℃, and even more preferably 30℃. The culture process of this invention is accompanied by oscillation, and the oscillation speed is preferably 150-250 rpm, more preferably 180-220 rpm, and even more preferably 200 rpm.

[0026] This invention also provides genes related to strain mismatch repair systems. MSH1 , MSH2 , MSH3 , MSH4 , MSH5 , MSH6 , MLH1 , MLH2, MLH3 or PMS1 Application in improving vanillin tolerance in brewer's yeast.

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] The *Saccharomyces cerevisiae* BY4741 used in this invention was purchased from Shanghai Weidi Biotechnology Co., Ltd., and *Escherichia coli* DH5α was purchased from Shanghai Bioengineering Co., Ltd. The *Saccharomyces cerevisiae* expression vectors p426-SNR52-gRNA, p426-SNR52-GGA, and p414-TEF2-Cas9 were purchased from Beijing Zhongyuan Heju Biotechnology Co., Ltd. Phusion high-fidelity DNA polymerase, T4 ligase, restriction endonucleases, plasmid extraction kits, DNA purification kits, gel extraction kits, and yeast genomic DNA extraction kits were all purchased from Shanghai Bioengineering Co., Ltd. The Taq HiFi PCR mix was purchased from Polymer Biotechnology Co., Ltd.

[0029] The LB medium consisted of 10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl, with double-distilled water added to bring the total volume to 1 L. The medium was then sterilized at 115°C under 0.1 MPa pressure for 30 min.

[0030] YPD culture medium consists of 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and double-distilled water to a final volume of 1 L. The medium is then sterilized at 115°C under 0.1 MPa pressure for 30 min.

[0031] The SC-LEU medium consists of: 6.7 g / L yeast nitrogen base, 1.4 g / L yeast auxotrophic medium supplement (without leucine), 20 g / L glucose, double-distilled water to a final volume of 1 L, and sterilized at 115°C under 0.1 MPa pressure for 20 min.

[0032] The SC-LEU-URA medium consists of: 6.7 g / L yeast nitrogen basal, 1.4 g / L yeast auxotrophic medium supplement (excluding leucine and uracil), 20 g / L glucose, double-distilled water to a final volume of 1 L, and sterilized at 115°C under 0.1 MPa pressure for 20 min.

[0033] The SC complete culture medium consists of: 6.7 g / L yeast nitrogen source basal, 1.4 g / L yeast auxotrophic medium supplement, 20 g / L glucose, double-distilled water to make up to 1 L, and sterilized at 115℃ under 0.1 MPa pressure for 20 min.

[0034] 100x 5-Fluorotic Acid: 100 mg of 5-fluoroorotic acid was dissolved in 1 mL using DMSO.

[0035] Example 1: Construction of strain RARE12

[0036] (a) Constructing a CRISPR gRNA expression module

[0037] 1. Constructing the p426-gRNA(AAD3) plasmid

[0038] Using the p426-SNR52-gRNA vector (addgene#43803) as a template, and primers F_gRNA.AAD3 (SEQ ID NO:1) and R_SUP4 (SEQ ID NO:23) as primers (Table 1), the gRNA (AAD3) fragment was amplified by PCR. The target band was recovered using a DNA purification kit to obtain the gel recovery product. PCR amplification conditions: 25 μL Taq HiFi PCR mix, 0.5 μL template, 1 μL each of upper and lower primers, and 22.5 μL double-distilled water; 98℃ for 2 min, 98℃ for 10 s, 56℃ for 30 s, 72℃ for 1 min, 30 cycles; 72℃ for 2 min. After digestion with BsaI for 4 h, the p426-SNR52-GGA plasmid was recovered by gel extraction, and then mixed with the gRNA (AAD3) fragment. Ligation was performed using T4 ligase and BsaI under the following conditions: 37℃ for 10 min; 37℃ for 10 min, 16℃ for 10 min, 4 cycles; 20℃ for 10 min. The ligated product was transformed into *E. coli* DH5α competent cells. Positive clones were obtained by verification using primers with nucleotide sequences such as SEQ ID NO:1 and SEQ ID NO:23. The gRNA expression plasmid p426-gRNA (AAD3) was extracted, and the sequencing results were completely consistent with the designed plasmid DNA sequence. 2.

[0040] As described above, construct the following plasmids respectively: p426-gRNA(ARI1), p426-gRNA(YPR1), p426-gRNA(YDR541C), p426-gRNA(GCY1), p426-gRNA(SFA1), p426-gRNA(YDL124W), p426-gRNA(GRE3), p426-gRNA(GRE2), p426-gRNA(ADH6), p426-gRNA(ADH7), and p426-gRNA(HFD1).The p426-gRNA (ARI1) plasmid was constructed using primers F_gRNA.ARI1 (as shown in SEQ ID NO:2) and R_SUP4 (as shown in SEQ ID NO:23); the p426-gRNA (YPR1) plasmid was constructed using primers F_gRNA.YPR1 (as shown in SEQ ID NO:3) and R_SUP4 (as shown in SEQ ID NO:23); the p426-gRNA (YDR541C) plasmid was constructed using primers F_gRNA.YDR541C (as shown in SEQ ID NO:4) and R_SUP4 (as shown in SEQ ID NO:23); the p426-gRNA (GCY1) plasmid was constructed using primers F_gRNA.GCY1 (as shown in SEQ ID NO:5) and R_SUP4 (as shown in SEQ ID NO:23); and the p426-gRNA (SFA1) plasmid was constructed using primers F_gRNA.GCY1 (as shown in SEQ ID NO:5) and R_SUP4 (as shown in SEQ ID NO:23). The primers for constructing the p426-gRNA (YDL124W) plasmid were F_gRNA.SFA1 as shown in SEQ ID NO:6 and R_SUP4 as shown in SEQ ID NO:23. The primers for constructing the p426-gRNA (GRE3) plasmid were F_gRNA.GRE3 as shown in SEQ ID NO:8 and R_SUP4 as shown in SEQ ID NO:23. The primers for constructing the p426-gRNA (GRE2) plasmid were F_gRNA.GRE2 as shown in SEQ ID NO:9 and R_SUP4 as shown in SEQ ID NO:23. The primers for constructing the p426-gRNA (ADH6) plasmid were F_gRNA.ADH6 as shown in SEQ ID NO:10 and R_SUP4 as shown in SEQ ID NO:23. Primer R_SUP4 as shown in NO:23 was used for constructing the p426-gRNA(ADH7) plasmid with primers F_gRNA.ADH7 as shown in SEQ ID NO:11 and R_SUP4 as shown in SEQ ID NO:23; primers F_gRNA.HFD1 as shown in SEQ ID NO:12 and R_SUP4 as shown in SEQ ID NO:23 were used for constructing the p426-gRNA(HFD1) plasmid. Specific primer sequence information is shown in Table 1.

[0041] (II) Preparation of targeted knockout fragments

[0042] Using F-AAD3-Del (nucleotide sequence shown in SEQ ID NO:24) and R-AAD3-Del (nucleotide sequence shown in SEQ ID NO:25) as primers (Table 1), the AAD3 knockout integration fragment was obtained by PCR amplification. The PCR amplification conditions were as follows: 25 μL of TaqHiFi PCR mix, 1 μL of template, 1 μL of upper and lower primers, and 22 μL of double-distilled water; 30 cycles of 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, and 72℃ for 15 s; and 72℃ for 1 min.

[0043] Using F-ARI1-Del (nucleotide sequence as shown in SEQ ID NO:26) and R-ARI1-Del (nucleotide sequence as shown in SEQ ID NO:27) as primers (Table 1), the ARI1 knockout integration fragment was obtained by PCR amplification. The PCR amplification conditions were as follows: 25 μL of TaqHiFi PCR mix, 1 μL of template, 1 μL of upper and lower primers, and 22 μL of double-distilled water; 30 cycles of 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, and 72℃ for 15 s; and 72℃ for 1 min.

[0044] Using F-YPR1-Del (nucleotide sequence shown in SEQ ID NO:28) and R-YPR1-Del (nucleotide sequence shown in SEQ ID NO:29) as primers (Table 1), the YPR1 knockout integration fragment was obtained by PCR amplification. The PCR amplification conditions were as follows: 25 μL of TaqHiFi PCR mix, 1 μL of template, 1 μL of upper and lower primers, and 22 μL of double-distilled water; 30 cycles of 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, and 72℃ for 15 s; and 72℃ for 1 min.

[0045] Using F-YDR541C-Del (nucleotide sequence as shown in SEQ ID NO:30) and R-YDR541C-Del (nucleotide sequence as shown in SEQ ID NO:31) as primers (Table 1), the YDR541C knockout integration fragment was obtained by PCR amplification. The PCR amplification conditions were as follows: 25 μL of Taq HiFi PCR mix, 1 μL of template, 1 μL of upper and lower primers, and 22 μL of double-distilled water; 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, 72℃ for 15 s, for 30 cycles; 72℃ for 1 min.

[0046] Using F-GCY1-Del (nucleotide sequence shown in SEQ ID NO:32) and R-GCY1-Del (nucleotide sequence shown in SEQ ID NO:33) as primers (Table 1), the GCY1 knockout integration fragment was obtained by PCR amplification. The PCR amplification conditions were as follows: 25 μL of TaqHiFi PCR mix, 1 μL of template, 1 μL of upper and lower primers, and 22 μL of double-distilled water; 30 cycles of 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, and 72℃ for 15 s; and 72℃ for 1 min.

[0047] Using F-SFA1-Del (nucleotide sequence as shown in SEQ ID NO:34) and R-SFA1-Del (nucleotide sequence as shown in SEQ ID NO:35) as primers (Table 1), the SFA1 knockout integration fragment was obtained by PCR amplification. The PCR amplification conditions were as follows: 25 μL of TaqHiFi PCR mix, 1 μL of template, 1 μL of upper and lower primers, and 22 μL of double-distilled water; 30 cycles of 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, and 72℃ for 15 s; and 72℃ for 1 min.

[0048] Using F-YDL124W-Del (nucleotide sequence shown in SEQ ID NO:36) and R-YDL124W-Del (nucleotide sequence shown in SEQ ID NO:37) as primers (Table 1), the YDL124W knockout integration fragment was obtained by PCR amplification. PCR amplification conditions: 25 μL Taq HiFi PCR mix, 1 μL template, 1 μL each of the upper and lower primers, and 22 μL double-distilled water; 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, 72℃ for 15 s, for 30 cycles; 72℃ for 1 min.

[0049] Using F-GRE3-Del (nucleotide sequence as shown in SEQ ID NO:38) and R-GRE3-Del (nucleotide sequence as shown in SEQ ID NO:39) as primers (Table 1), the GRE3 knockout integration fragment was obtained by PCR amplification. The PCR amplification conditions were as follows: 25 μL of TaqHiFi PCR mix, 1 μL of template, 1 μL of upper and lower primers, and 22 μL of double-distilled water; 30 cycles of 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, and 72℃ for 15 s; and 72℃ for 1 min.

[0050] Using F-GRE2-Del (nucleotide sequence as shown in SEQ ID NO:40) and R-GRE2-Del (nucleotide sequence as shown in SEQ ID NO:41) as primers (Table 1), the GRE2 knockout integration fragment was obtained by PCR amplification. The PCR amplification conditions were as follows: 25 μL of TaqHiFi PCR mix, 1 μL of template, 1 μL of upper and lower primers, and 22 μL of double-distilled water; 30 cycles of 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, and 72℃ for 15 s; and 72℃ for 1 min.

[0051] Using F-ADH6-Del (nucleotide sequence shown in SEQ ID NO:42) and R-ADH6-Del (nucleotide sequence shown in SEQ ID NO:43) as primers (Table 1), the ADH6 knockout integration fragment was obtained by PCR amplification. The PCR amplification conditions were as follows: 25 μL of TaqHiFi PCR mix, 1 μL of template, 1 μL of upper and lower primers, and 22 μL of double-distilled water; 30 cycles of 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, and 72℃ for 15 s; and 72℃ for 1 min.

[0052] Using F-ADH7-Del (nucleotide sequence shown in SEQ ID NO:44) and R-ADH7-Del (nucleotide sequence shown in SEQ ID NO:45) as primers (Table 1), the ADH7 knockout integration fragment was obtained by PCR amplification. The PCR amplification conditions were as follows: 25 μL of TaqHiFi PCR mix, 1 μL of template, 1 μL of upper and lower primers, and 22 μL of double-distilled water; 30 cycles of 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, and 72℃ for 15 s; and 72℃ for 1 min.

[0053] Using F-HFD1-Del (nucleotide sequence shown in SEQ ID NO:46) and R-HFD1-Del (nucleotide sequence shown in SEQ ID NO:47) as primers (Table 1), the HFD1 knockout integration fragment was obtained by PCR amplification. The PCR amplification conditions were as follows: 25 μL of TaqHiFi PCR mix, 1 μL of template, 1 μL of upper and lower primers, and 22 μL of double-distilled water; 30 cycles of 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, and 72℃ for 15 s; and 72℃ for 1 min.

[0054] (III) Construction of Saccharomyces cerevisiae strain RARE12

[0055] Competent cells of *Saccharomyces cerevisiae* BY4741 were prepared and transformed with the p415-TEF2-Cas9 plasmid. The cells were cultured on SC-LEU plates at 30°C for 2-4 days, and the resulting single colonies were named *Saccharomyces cerevisiae* Y1. Competent cells of Y1 were then prepared, and the obtained p426-gRNA (AAD3) plasmid and the obtained AAD3 knockout integrated fragment were transformed into Y1 competent cells. The cells were cultured on SC-LEU-URA plates at 30°C for 2-4 days. Single colonies were streaked onto SC-LEU-URA solid plates, and PCR verification was performed using primers SEQ ID NO:68 and SEQ ID NO:69. Subsequently, the p426-gRNA (AAD3) plasmid was removed from the cells by streaking onto SC-LEU plates containing 1 mg / mL 5-fluoroorotic acid. The correctly identified *Saccharomyces cerevisiae* strain was named recombinant strain RARE1.

[0056] The plasmid p426-gRNA (ARI1) and the ARI1 knockout integration fragment were transformed into the above-mentioned RARE1 competent cells and cultured on SC-LEU-URA plates at 30°C for 2-4 days. Single colonies were streaked onto SC-LEU-URA solid plates and verified by PCR using primers SEQ ID NO:70 and SEQ ID NO:71. Subsequently, the p426-gRNA (ARI1) plasmid was removed from the cells by streaking onto SC-LEU plates containing 1 mg / mL of 5-fluoroorotic acid. The correct Saccharomyces cerevisiae strain was named recombinant strain RARE2.

[0057] The plasmid p426-gRNA (YPR1) and the YPR1 knockout integration fragment were transformed into the above-mentioned RARE2 competent cells and cultured on SC-LEU-URA plates at 30°C for 2-4 days. Single colonies were streaked onto SC-LEU-URA solid plates and verified by PCR using primers SEQ ID NO:72 and SEQ ID NO:73. Subsequently, the cells were streaked onto SC-LEU plates containing 1 mg / mL of 5-fluoroorotic acid to remove the intracellular p426-gRNA (YPR1) plasmid. The correct Saccharomyces cerevisiae strain was named recombinant strain RARE3.

[0058] The plasmid p426-gRNA (YDR541C) and the YDR541C knockout integration fragment were transformed into the above-mentioned RARE3 competent cells and cultured on SC-LEU-URA plates at 30°C for 2-4 days. Single colonies were streaked onto SC-LEU-URA solid plates and verified by PCR using primers SEQ ID NO:74 and SEQ ID NO:75. Subsequently, the p426-gRNA (YDR541C) plasmid was removed from the cells by streaking onto SC-LEU plates containing 1 mg / mL of 5-fluoroorotic acid. The correct Saccharomyces cerevisiae strain was named recombinant strain RARE4.

[0059] The plasmid p426-gRNA (GCY1) and the GCY1 knockout integration fragment were transformed into the above-mentioned RARE4 competent cells and cultured on SC-LEU-URA plates at 30°C for 2-4 days. Single colonies were streaked onto SC-LEU-URA solid plates and verified by PCR using primers SEQ ID NO:76 and SEQ ID NO:77. Subsequently, the cells were streaked onto SC-LEU plates containing 1 mg / mL of 5-fluoroorotic acid to remove the intracellular p426-gRNA (GCY1) plasmid. The correct Saccharomyces cerevisiae strain was named recombinant strain RARE5.

[0060] The plasmid p426-gRNA (SFA1) and the SFA1 knockout integration fragment were transformed into the above-mentioned RARE5 competent cells and cultured on SC-LEU-URA plates at 30°C for 2-4 days. Single colonies were streaked onto SC-LEU-URA solid plates and verified by PCR using primers SEQ ID NO:78 and SEQ ID NO:79. Subsequently, the cells were streaked onto SC-LEU plates containing 1 mg / mL of 5-fluoroorotic acid to remove the intracellular p426-gRNA (SFA1) plasmid. The correct Saccharomyces cerevisiae strain was named recombinant strain RARE6.

[0061] The plasmid p426-gRNA (YDL124W) and the YDL124W knockout integration fragment were transformed into the above-mentioned RARE6 competent cells and cultured on SC-LEU-URA plates at 30°C for 2-4 days. Single colonies were streaked onto SC-LEU-URA solid plates and verified by PCR using primers SEQ ID NO:80 and SEQ ID NO:81. Subsequently, the p426-gRNA (YDL124W) plasmid was removed from the cells by streaking onto SC-LEU plates containing 1 mg / mL of 5-fluoroorotic acid. The correct Saccharomyces cerevisiae strain was named recombinant strain RARE7.

[0062] The plasmid p426-gRNA (GRE3) and the GRE3 knockout integration fragment were transformed into the above-mentioned RARE7 competent cells and cultured on SC-LEU-URA plates at 30°C for 2-4 days. Single colonies were streaked onto SC-LEU-URA solid plates and verified by PCR using primers SEQ ID NO:82 and SEQ ID NO:83. Subsequently, the p426-gRNA (GRE3) plasmid was removed from the cells by streaking onto SC-LEU plates containing 1 mg / mL of 5-fluoroorotic acid. The correct Saccharomyces cerevisiae strain was named recombinant strain RARE8.

[0063] The plasmid p426-gRNA (GRE2) and the GRE2 knockout integration fragment were transformed into the above-mentioned RARE8 competent cells and cultured on SC-LEU-URA plates at 30°C for 2-4 days. Single colonies were streaked onto SC-LEU-URA solid plates and verified by PCR using primers SEQ ID NO:84 and SEQ ID NO:85. Subsequently, the cells were streaked onto SC-LEU plates containing 1 mg / mL of 5-fluoroorotic acid to remove the intracellular p426-gRNA (GRE2) plasmid. The correct Saccharomyces cerevisiae strain was named recombinant strain RARE9.

[0064] The plasmid p426-gRNA (ADH6) and the ADH6 knockout integration fragment were transformed into the above-mentioned RARE9 competent cells and cultured on SC-LEU-URA plates at 30°C for 2-4 days. Single colonies were streaked onto SC-LEU-URA solid plates and verified by PCR using primers SEQ ID NO:86 and SEQ ID NO:87. Subsequently, the cells were streaked onto SC-LEU plates containing 1 mg / mL of 5-fluoroorotic acid to remove the intracellular p426-gRNA (ADH6) plasmid. The correct Saccharomyces cerevisiae strain was named recombinant strain RARE10.

[0065] The plasmid p426-gRNA (ADH7) and the ADH7 knockout integration fragment were transformed into the above-mentioned RARE10 competent cells and cultured on SC-LEU-URA plates at 30°C for 2-4 days. Single colonies were streaked onto SC-LEU-URA solid plates and verified by PCR using primers SEQ ID NO:88 and SEQ ID NO:89. Subsequently, the plasmid p426-gRNA (ADH7) was removed from the cells by streaking onto SC-LEU plates containing 1 mg / mL of 5-fluoroorotic acid. The correct Saccharomyces cerevisiae strain was named the recombinant strain RARE11.

[0066] The plasmid p426-gRNA (HFD1) and the HFD1 knockout integration fragment were transformed into the above-mentioned RARE-11 competent cells and cultured on SC-LEU-URA plates at 30°C for 2-4 days. Single colonies were streaked onto SC-LEU-URA solid plates and verified by PCR using primers SEQ ID NO:90 and SEQ ID NO:91. Subsequently, the p426-gRNA (HFD1) plasmid was removed from the cells by streaking onto SC-LEU plates containing 1 mg / mL of 5-fluoroorotic acid. The correct Saccharomyces cerevisiae strain was named recombinant strain RARE12.

[0067] Table 1 Primers used for PCR amplification and their corresponding sequence information.

[0068] Example 2: Construction of engineered brewer's yeast

[0069] (a) Constructing a CRISPR gRNA expression module

[0070] 1. Constructing the p426-gRNA(MSH1) plasmid

[0071] Using the p426-SNR52-gRNA vector (addgene#43803) as a template, and primers F_gRNA.MSH1 (SEQ ID NO:13) and R_SUP4 (SEQ ID NO:23) as primers (Table 1), the gRNA (MSH1) fragment was amplified by PCR. The target band was recovered using a DNA purification kit to obtain the gel recovery product. PCR amplification conditions: 25 μL Taq HiFi PCR mix, 0.5 μL template, 1 μL each of upper and lower primers, and 22.5 μL double-distilled water; 98℃ for 2 min, 98℃ for 10 s, 56℃ for 30 s, 72℃ for 1 min, 30 cycles; 72℃ for 2 min. After digestion with BsaI for 4 h, the p426-SNR52-GGA plasmid was recovered by gel electrophoresis, and then mixed with the gRNA (MSH1) fragment. Ligation was performed using T4 ligase and BsaI under the following conditions: 37℃ for 10 min; 37℃ for 10 min, 16℃ for 10 min, 4 cycles; 20℃ for 10 min. The ligated product was transformed into *E. coli* DH5α competent cells. Positive clones were obtained by verification with primers containing nucleotide sequences such as SEQ ID NO:13 and SEQ ID NO:23. The gRNA expression plasmid p426-gRNA (MSH1) was extracted, and the sequencing results were completely consistent with the designed plasmid DNA sequence. 2.

[0073] Similar to the above operations, construct the p426-gRNA(MSH2), p426-gRNA(MSH3), p426-gRNA(MSH4), p426-gRNA(MSH5), p426-gRNA(MSH6), p426-gRNA(MLH1), p426-gRNA(MLH2), p426-gRNA(MLH3), or p426-gRNA(PMS1) plasmids respectively. The p426-gRNA (MSH2) plasmid was constructed using primers F_gRNA.MSH2 (as shown in SEQ ID NO:14) and R_SUP4 (as shown in SEQ ID NO:23); the p426-gRNA (MSH3) plasmid was constructed using primers F_gRNA.MSH3 (as shown in SEQ ID NO:15) and R_SUP4 (as shown in SEQ ID NO:23); the p426-gRNA (MSH4) plasmid was constructed using primers F_gRNA.MSH4 (as shown in SEQ ID NO:16) and R_SUP4 (as shown in SEQ ID NO:23); the p426-gRNA (MSH5) plasmid was constructed using primers F_gRNA.MSH5 (as shown in SEQ ID NO:17) and R_SUP4 (as shown in SEQ ID NO:23); and the p426-gRNA (MSH6) plasmid was constructed using primers F_gRNA.MSH5 (as shown in SEQ ID NO:17) and R_SUP4 (as shown in SEQ ID NO:23). The primers for constructing the p426-gRNA (MLH1) plasmid were F_gRNA.MSH6 as shown in SEQ ID NO:18 and R_SUP4 as shown in SEQ ID NO:23; the primers for constructing the p426-gRNA (MLH2) plasmid were F_gRNA.MLH1 as shown in SEQ ID NO:19 and R_SUP4 as shown in SEQ ID NO:23; the primers for constructing the p426-gRNA (MLH3) plasmid were F_gRNA.MLH3 as shown in SEQ ID NO:21 and R_SUP4 as shown in SEQ ID NO:23; and the primers for constructing the p426-gRNA (PMS1) plasmid were F_gRNA.PMS1 as shown in SEQ ID NO:22 and R_SUP4 as shown in SEQ ID NO:23. NO:23 shows R_SUP4 as the primer. See Table 1 for specific primer sequence information.

[0074] (II) Preparation of targeted knockout fragments

[0075] Using F-MSH1-Del (nucleotide sequence as shown in SEQ ID NO:48) and R-MSH1-Del (nucleotide sequence as shown in SEQ ID NO:49) as primers (Table 1), the MSH1 knockout integration fragment was obtained by PCR amplification. ΔMSH1 PCR amplification conditions: 25 μL Taq HiFi PCR mix, 0.5 μL template, 1 μL each of upper and lower primers, and 22.5 μL double-distilled water; 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, 72℃ for 15 s, for 30 cycles; 72℃ for 1 min.

[0076] Using F-MSH2-Del (nucleotide sequence as shown in SEQ ID NO:50) and R-MSH2-Del (nucleotide sequence as shown in SEQ ID NO:51) as primers (Table 1), the MSH2 knockout integration fragment was obtained by PCR amplification. ΔMSH2 PCR amplification conditions: 25 μL Taq HiFi PCR mix, 0.5 μL template, 1 μL each of upper and lower primers, and 22.5 μL double-distilled water; 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, 72℃ for 15 s, for 30 cycles; 72℃ for 1 min.

[0077] Using F-MSH3-Del (nucleotide sequence as shown in SEQ ID NO:52) and R-MSH3-Del (nucleotide sequence as shown in SEQ ID NO:53) as primers (Table 1), the MSH3 knockout integration fragment was obtained by PCR amplification. ΔMSH3 PCR amplification conditions: 25 μL Taq HiFi PCR mix, 0.5 μL template, 1 μL each of upper and lower primers, and 22.5 μL double-distilled water; 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, 72℃ for 15 s, for 30 cycles; 72℃ for 1 min.

[0078] Using F-MSH4-Del (nucleotide sequence as shown in SEQ ID NO:54) and R-MSH4-Del (nucleotide sequence as shown in SEQ ID NO:55) as primers (Table 1), the MSH4 knockout integration fragment was obtained by PCR amplification. ΔMSH4 PCR amplification conditions: 25 μL Taq HiFi PCR mix, 0.5 μL template, 1 μL each of upper and lower primers, and 22.5 μL double-distilled water; 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, 72℃ for 15 s, for 30 cycles; 72℃ for 1 min.

[0079] Using F-MSH5-Del (nucleotide sequence as shown in SEQ ID NO:56) and R-MSH5-Del (nucleotide sequence as shown in SEQ ID NO:57) as primers (Table 1), the MSH5 knockout integration fragment was obtained by PCR amplification. ΔMSH5 PCR amplification conditions: 25 μL Taq HiFi PCR mix, 0.5 μL template, 1 μL each of upper and lower primers, and 22.5 μL double-distilled water; 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, 72℃ for 15 s, for 30 cycles; 72℃ for 1 min.

[0080] Using F-MSH6-Del (nucleotide sequence as shown in SEQ ID NO:58) and R-MSH6-Del (nucleotide sequence as shown in SEQ ID NO:59) as primers (Table 1), the MSH6 knockout integration fragment was obtained by PCR amplification. ΔMSH6 PCR amplification conditions: 25 μL Taq HiFi PCR mix, 0.5 μL template, 1 μL each of upper and lower primers, and 22.5 μL double-distilled water; 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, 72℃ for 15 s, for 30 cycles; 72℃ for 1 min.

[0081] Using F-MLH1-Del (nucleotide sequence as shown in SEQ ID NO:60) and R-MLH1-Del (nucleotide sequence as shown in SEQ ID NO:61) as primers (Table 1), the MLH1 knockout integration fragment was obtained by PCR amplification. ΔMLH1 PCR amplification conditions: 25 μL Taq HiFi PCR mix, 0.5 μL template, 1 μL each of upper and lower primers, and 22.5 μL double-distilled water; 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, 72℃ for 15 s, for 30 cycles; 72℃ for 1 min.

[0082] Using F-MLH2-Del (nucleotide sequence as shown in SEQ ID NO:62) and R-MLH2-Del (nucleotide sequence as shown in SEQ ID NO:63) as primers (Table 1), the MLH2 knockout integration fragment was obtained by PCR amplification. ΔMLH2 PCR amplification conditions: 25 μL Taq HiFi PCR mix, 0.5 μL template, 1 μL each of upper and lower primers, and 22.5 μL double-distilled water; 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, 72℃ for 15 s, for 30 cycles; 72℃ for 1 min.

[0083] Using F-MLH3-Del (nucleotide sequence as shown in SEQ ID NO:64) and R-MLH3-Del (nucleotide sequence as shown in SEQ ID NO:65) as primers (Table 1), the MLH3 knockout integration fragment was obtained by PCR amplification. ΔMLH3 PCR amplification conditions: 25 μL Taq HiFi PCR mix, 0.5 μL template, 1 μL each of upper and lower primers, and 22.5 μL double-distilled water; 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, 72℃ for 15 s, for 30 cycles; 72℃ for 1 min.

[0084] Using F-PMS1-Del (nucleotide sequence as shown in SEQ ID NO:66) and R-PMS1-Del (nucleotide sequence as shown in SEQ ID NO:67) as primers (Table 1), the PMS1 knockout integration fragment was obtained by PCR amplification. ΔPMS1 PCR amplification conditions: 25 μL Taq HiFi PCR mix, 0.5 μL template, 1 μL each of upper and lower primers, and 22.5 μL double-distilled water; 94℃ for 2 min, 94℃ for 15 s, 50℃ for 15 s, 72℃ for 15 s, for 30 cycles; 72℃ for 1 min.

[0085] (III) Construction of engineered Saccharomyces cerevisiae (mismatch repair gene knockout strain)

[0086] The p426-gRNA (MSH1) plasmid and the MSH1 knockout integration fragment were transformed into Cas9-integrated Saccharomyces cerevisiae RARE12 competent cells using electroporation. The cells were cultured on SC-LEU-URA plates at 30°C for 2-4 days. Single colonies were then streaked onto SC-LEU-URA plates and verified by PCR using primers SEQ ID NO:92 and SEQ ID NO:93. Subsequently, the cells were streaked onto SC-LEU plates containing 1 mg / mL 5-fluoroorotic acid to remove the intracellular p426-gRNA (MSH1) plasmid. The correctly identified Saccharomyces cerevisiae strain was named the engineered strain RARE12. ΔMSH1 ).

[0087] The plasmid p426-gRNA (MSH2) and the MSH2 knockout integration fragment were transformed into Cas9-integrated Saccharomyces cerevisiae RARE12 competent cells using electroporation. The cells were cultured on SC-LEU-URA plates at 30°C for 2-4 days. Single colonies were streaked onto SC-LEU-URA solid plates and verified by PCR using primers SEQ ID NO:94 and SEQ ID NO:95. Subsequently, the cells were streaked onto SC-LEU plates containing 1 mg / mL of 5-fluoroorotic acid to remove the intracellular p426-gRNA (MSH2) plasmid. The correct Saccharomyces cerevisiae strain was named the engineered strain RARE12 (ΔMSH2).

[0088] Similar to the above operations, engineered strain RARE12 was constructed. ΔMSH3 ), RARE12 ΔMSH4 ), RARE12 ΔMSH5 ), RARE12 ΔMSH6 ), RARE12 ΔMLH1 ), RARE12 ΔMLH2 ), RARE12 ΔMLH3 ) or RARE12 ( ΔPMS1 Among them, the engineered strain RARE12 was constructed. ΔMSH3 The primers involved are the sequences shown in SEQ ID NO:96 and SEQ ID NO:97; the engineered strain RARE12 was constructed. ΔMSH4 The primers involved are the sequences shown in SEQ ID NO:98 and SEQ ID NO:99; the engineered strain RARE12 was constructed. ΔMSH5 The primers involved are the sequences shown in SEQ ID NO:100 and SEQ ID NO:101; the engineered strain RARE12 was constructed. Δ MSH6 The primers involved are the sequences shown in SEQ ID NO:102 and SEQ ID NO:103; the engineered strain RARE12 was constructed. ΔMLH1 The primers involved are the sequences shown in SEQ ID NO:104 and SEQ ID NO:105; the engineered strain RARE12 was constructed. ΔMLH2 The primers involved are the sequences shown in SEQ ID NO:106 and SEQ ID NO:107; the engineered strain RARE12 was constructed. ΔMLH3 The primers involved are the sequences shown in SEQ ID NO:108 and SEQ ID NO:109; the engineered strain RARE12 was constructed.ΔPMS1 The primers involved are the sequences shown in SEQ ID NO:110 and SEQ ID NO:111.

[0089] Example 3: Preparation of vanillin-tolerant recombinant Saccharomyces cerevisiae strain

[0090] The above-mentioned engineered strain RARE12 ( ΔMSH1 ), RARE12 ΔMSH2 ), RARE12 ΔMSH3 ), RARE12 Δ MSH4 ), RARE12 ΔMSH5 ), RARE12 ΔMSH6 ), RARE12 ΔMLH1 ), RARE12 ΔMLH2 ), RARE12 Δ MLH3 ), RARE12 ΔPMS1 Single clones, about the size of a grain of rice, were inoculated into SC-LEU medium to prepare seed culture. The seed culture was then transferred to SC-LEU medium containing 0.5 g / L vanillin and cultured aerobically with shaking at 200 rpm and 30°C until the stationary phase, yielding the first-generation culture. The first-generation culture was then transferred to SC-LEU medium containing 0.75 g / L vanillin and cultured under the same conditions to obtain the second-generation culture. The previous generation cultures were then progressively transferred to the corresponding SC-LEU medium at vanillin concentration gradients of 1.0, 1.2, 1.3, 1.4, 1.5, 1.6, and 1.7 g / L, each time cultured aerobically until the stationary phase, yielding the third to ninth generations of cultures, thus obtaining the vanillin-tolerant recombinant Saccharomyces cerevisiae strains.

[0091] The stationary period referred to in this experiment was defined as: sampling every 12 hours to measure the bacterial biomass (OD). 600 In two measurements taken 12 hours apart, OD 600 The difference does not exceed 3.

[0092] (a) Growth curves of culture media with different vanillin concentrations

[0093] The engineered strain RARE12 ( ΔMSH1 ), RARE12 ΔMSH2 ), RARE12 ΔMSH3 ), RARE12 Δ MSH ), RARE12 ​ ), RARE12 ​ ), RARE12 ​), RARE12 ​ ), RARE12 ​ ​ ), RARE12 ​ The strains RARE12 and the starting strain were inoculated into shake flasks containing 0.5 g / L, 0.75 g / L, 1.3 g / L, and 1.7 g / L vanillin, respectively, and cultured under aerobic conditions with shaking at 200 rpm at 30°C. Samples were taken every 12 h to determine the bacterial biomass (OD). 600 ), and plot the bacterial growth curve.

[0094] The results are as follows ​ As shown, in 0.5 g / L vanillin medium, RARE12 ( ​ ), RARE12 ​ ), RARE12 ​ ), RARE12 ​ ), RARE12 ​ ), RARE12 ​ ), RARE12 ​ ), RARE12 ​ ), RARE12 ​ The growth rates of the ) are basically the same, OD 600 Reaching 1.0 or higher, RARE12 ( ​ ​ RARE12 and vanillin were initially inhibited. In 1.0 g / L vanillin medium, RARE12 ( ​ ), RARE12 ​ ​ ), RARE12 ​ ), RARE12 ​ ), RARE12 ​ ), RARE12 ​ ), RARE12 ​ ​ ), RARE12 ​ ), RARE12 ​ ) can grow effectively, RARE12 ( ​ RARE12 and RARE12 could no longer grow. In 1.7 g / L vanillin medium, RARE12 ( ΔMSH2 ), RARE12 ΔMLH1 ), RARE12 ΔMLH3 OD of three strains 600 It can reach above 0.4. It is evident that the modified RARE12 ( ΔMSH2 ), RARE12 ΔMLH1 ), RARE12 ΔMLH3 It significantly improved tolerance to vanillin, achieving effective vanillin domestication.

[0095] In summary, the technical solution of this invention breaks through the traditional approach of metabolic engineering modification, and innovatively knocks out genes in the DNA mismatch repair system (…). MSH1 , MSH2 , MSH3 , MSH4 , MSH5 , MSH6 , MLH1 , MLH2 , MLH3 , PMS1 This improved the adaptive evolutionary ability of the strains and screened for mutants with significantly enhanced tolerance. Among them, Saccharomyces cerevisiae RARE12 ( ΔMLH1 The modified strain under 1.3 g / L vanillin stress had an OD of 600 The highest value reached 1.0, while the control group could not grow. Using this domestication method, the brewer's yeast RARE12 ( ΔMLH1 It can tolerate vanillin concentrations up to 1.7 g / L. This method can overcome the yield problem caused by vanillin toxicity and lay an effective foundation for the biosynthesis of high-concentration vanillin.

[0096] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant strain of *Saccharomyces cerevisiae* that enhances vanillin tolerance, characterized in that, Using strain RARE12 as the original strain, the mismatch repair genes MSH1, MSH2, MSH3, MSH4, MSH5, MSH6, MLH1, MLH2, MLH3 or PMS1 of strain RARE12 were knocked out, and then the strain was domesticated in a concentration gradient in a vanillin-containing medium to obtain the engineered strain.

2. The recombinant Saccharomyces cerevisiae strain according to claim 1, characterized in that, The strain RARE12 is obtained by sequentially knocking out the aldehyde accumulation genes AAD3, ARI1, YPR1, YDR541C, GCY1, SFA1, YDL124W, GRE3, GRE2, ADH6, ADH7, and HFD1 of Saccharomyces cerevisiae BY4741 as the original strain.

3. The recombinant Saccharomyces cerevisiae strain according to claim 1, characterized in that, The concentration gradient of vanillin is 0.5-1.7 g / L.

4. The method for constructing the recombinant Saccharomyces cerevisiae strain with enhanced vanillin tolerance according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Using p426-SNR52-gRNA as a template, gRNA expression fragments of genes MSH1, MSH2, MSH3, MSH4, MSH5, MSH6, MLH1, MLH2, MLH3 or PMS1 were amplified by PCR, and then ligated to the enzyme-digested vector plasmids to construct recombinant plasmids p426-gRNA(MSH1), p426-gRNA(MSH2), p426-gRNA(MSH3), p426-gRNA(MSH4), p426-gRNA(MSH5), p426-gRNA(MSH6), p426-gRNA(MLH1), p426-gRNA(MLH2), p426-gRNA(MLH3) or p426-gRNA(PMS1); (2) Transform the recombinant plasmid described in step (1) and the knockout integration fragment of the corresponding gene obtained by PCR amplification into RARE12 competent cells to obtain engineered strains RARE12(ΔMSH1), RARE12(ΔMSH2), RARE12(ΔMSH3), RARE12(ΔMSH4), RARE12(ΔMSH5), RARE12(ΔMSH6), RARE12(ΔMLH1), RARE12(ΔMLH2), RARE12(ΔMLH3) or RARE12(ΔPMS1); (3) The bacterial culture of the engineered strain obtained in step (2) is inoculated into a culture medium containing 0.5 g / L vanillin to obtain the first generation culture. Then, the previous generation culture is transferred sequentially according to the concentration gradient of 0.75-1.7 g / L vanillin to the ninth generation culture, which is obtained.

5. The construction method according to claim 4, characterized in that, The PCR amplification primers for the MSH1 gene in step (1) are F_gRNA.MSH1 as shown in SEQ ID NO:13 and R_SUP4 as shown in SEQ ID NO:23; The PCR amplification primers for the MSH2 gene are F_gRNA.MSH2 as shown in SEQ ID NO:14 and R_SUP4 as shown in SEQ ID NO:23; The PCR amplification primers for the MSH3 gene are F_gRNA.MSH3 as shown in SEQ ID NO:15 and R_SUP4 as shown in SEQ ID NO:23; The PCR amplification primers for the MSH4 gene are F_gRNA.MSH4 as shown in SEQ ID NO:16 and R_SUP4 as shown in SEQ ID NO:23; The PCR amplification primers for the MSH5 gene are F_gRNA.MSH5 as shown in SEQ ID NO:17 and R_SUP4 as shown in SEQ ID NO:23; The PCR amplification primers for the MSH6 gene are F_gRNA.MSH6 as shown in SEQ ID NO:18 and R_SUP4 as shown in SEQ ID NO:23; The PCR amplification primers for the MLH1 gene are F_gRNA.MLH1 as shown in SEQ ID NO:19 and R_SUP4 as shown in SEQ ID NO:23; The PCR amplification primers for the MLH2 gene are F_gRNA.MLH2 as shown in SEQ ID NO:20 and R_SUP4 as shown in SEQ ID NO:23; The PCR amplification primers for the MLH3 gene are F_gRNA.MLH3 as shown in SEQ ID NO:21 and R_SUP4 as shown in SEQ ID NO:23; The PCR amplification primers for the PMS1 gene are F_gRNA.PMS1 as shown in SEQ ID NO:22 and R_SUP4 as shown in SEQ ID NO:

23.

6. The construction method according to claim 4, characterized in that, Step (2) The PCR amplification primers for the MSH1 knockout integration fragment are F-MSH1-Del as shown in SEQ ID NO:48 and R-MSH1-Del as shown in SEQ ID NO:49; The PCR amplification primers for the MSH2 knockout integration fragment are F-MSH2-Del as shown in SEQ ID NO:50 and R-MSH2-Del as shown in SEQ ID NO:51; The PCR amplification primers for the MSH3 knockout integration fragment are F-MSH3-Del as shown in SEQ ID NO:52 and R-MSH3-Del as shown in SEQ ID NO:53; The PCR amplification primers for the MSH4 knockout integration fragment are F-MSH4-Del as shown in SEQ ID NO:54 and R-MSH4-Del as shown in SEQ ID NO:55; The PCR amplification primers for the MSH5 knockout integration fragment are F-MSH5-Del as shown in SEQ ID NO:56 and R-MSH5-Del as shown in SEQ ID NO:57; The PCR amplification primers for the MSH6 knockout integration fragment are F-MSH6-Del as shown in SEQ ID NO:58 and R-MSH6-Del as shown in SEQ ID NO:59; The PCR amplification primers for the MLH1 knockout integration fragment are F-MLH1-Del as shown in SEQ ID NO:60 and R-MLH1-Del as shown in SEQ ID NO:61; The PCR amplification primers for the MLH2 gene knockout integration fragment are F-MLH2-Del as shown in SEQ ID NO:62 and R-MLH2-Del as shown in SEQ ID NO:63; The PCR amplification primers for the MLH3 knockout integration fragment are F-MLH3-Del as shown in SEQ ID NO:64 and R-MLH3-Del as shown in SEQ ID NO:65; The PCR amplification primers for the PMS1 gene knockout integration fragment are F-PMS1-Del as shown in SEQ ID NO:66 and R-PMS1-Del as shown in SEQ ID NO:

67.

7. The construction method according to claim 4, characterized in that, The culture medium in step (3) consists of 6.5-7 g / L yeast nitrogen source base, 1.2-1.6 g / L yeast auxotrophic culture medium supplement and 18-22 g / L glucose.

8. The construction method according to claim 4, characterized in that, The amount of inoculation described in step (3) is 5-10% v / v.

9. The construction method according to claim 4, characterized in that, The culture temperature in step (3) is 28-32℃.

10. Application of strain mismatch repair system related genes MSH1, MSH2, MSH3, MSH4, MSH5, MSH6, MLH1, MLH2, MLH3 or PMS1 in improving vanillin tolerance in Saccharomyces cerevisiae.

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