An antioxidant stress saccharomyces cerevisiae engineering strain, a construction method and application thereof

Abstract

The application discloses an antioxidant stress Saccharomyces cerevisiae engineering strain and a construction method and application thereof. The Saccharomyces cerevisiae engineering strain has the following characteristics: a gene BIO5 is knocked out, and a gene HMO1 is overexpressed. The yeast gene engineering strain has significantly improved antioxidant stress resistance and recombinant protein production capacity. This is conducive to improving the efficiency of using Saccharomyces cerevisiae to produce recombinant proteins in industry and bringing higher economic benefits. In addition, the engineering strain has certain reference significance for designing and constructing strains resistant to other stresses and high-yield protein strains, and even certain reference significance for other researches such as treatment, prevention and drug screening platform construction of related diseases. The application has good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an engineered strain of Saccharomyces cerevisiae that is resistant to oxidative stress, its construction method, and its application. Background Technology

[0002] Saccharomyces cerevisiae is widely used in the production of fuels, chemicals, and various proteins with medical or industrial applications. It plays a crucial role in recombinant protein production. Currently, researchers have conducted extensive studies on the protein synthesis and secretion pathways of Saccharomyces cerevisiae. During protein synthesis, changes in the endoplasmic reticulum (ER) folding environment and folding requirements can induce ER stress and oxidative stress. Studies have shown that these two types of stress can significantly impact normal cellular physiological and metabolic activities. Severe stress can even lead to apoptosis when the expression requirements of recombinant proteins cannot be met. Therefore, enhancing the oxidative stress resistance of strains helps improve their ability to express recombinant proteins. Furthermore, ER stress and oxidative stress are closely related to many human diseases.

[0003] Chlamycin (Tm) and dithiothreitol (DTT) are two commonly used agents for inducing endoplasmic reticulum stress. Tm inhibits protein glycosylation, while DTT interferes with disulfide bond formation and has a certain cytotoxic effect. Inducing endoplasmic reticulum stress in cells is usually accompanied by oxidative stress, thereby disrupting the redox balance of the endoplasmic reticulum. By inducing oxidative stress in yeast cells using Tm and DTT, and testing the antioxidant capacity of modified strains, key gene targets for oxidative stress resistance can be identified. This is of great significance for optimizing recombinant protein production strategies. Furthermore, this invention also has certain reference value for research on the treatment, prevention, and drug screening platform construction of related diseases. Summary of the Invention

[0004] This invention aims to address the problem of limited protein yield due to oxidative stress on cells during the production of recombinant proteins using *Saccharomyces cerevisiae*. To this end, it provides an improved *Saccharomyces cerevisiae* engineered strain and its construction method to enhance its oxidative stress resistance and optimize recombinant protein production. This invention identifies the BIO5 and HMO1 genes as key genes and combines them in a synergistic manner. Specifically, gene editing technology is used to knock out gene BIO5 and overexpress gene HMO1. Oxidative stress tests show that the *Saccharomyces cerevisiae* strain exhibits significantly improved oxidative stress tolerance and a significantly increased recombinant protein yield. This invention provides application potential for constructing a high-protein-secreting, stress-resistant *Saccharomyces cerevisiae* strain.

[0005] The primary objective of this invention is to provide an engineered strain of Saccharomyces cerevisiae that is resistant to oxidative stress.

[0006] Another object of the present invention is to provide a method for constructing the above-mentioned engineered strain of Saccharomyces cerevisiae with antioxidant stress.

[0007] Another object of the present invention is to provide the application of the above-mentioned engineered strain of Saccharomyces cerevisiae that is resistant to oxidative stress.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] An engineered strain of Saccharomyces cerevisiae resistant to oxidative stress has the following characteristics: the BIO5 gene is knocked out and the HMO1 gene is overexpressed.

[0010] The starting strain of the engineered Saccharomyces cerevisiae is CEN.PK background Saccharomyces cerevisiae, which is a commonly used yeast strain and is widely used in metabolic engineering and systems biology research in industry and academia (Microb Cell Fact, 2012, 11, 36), and has good representativeness and universality.

[0011] The preferred starting strain is a Saccharomyces cerevisiae strain that expresses recombinant proteins by introducing a plasmid expressing recombinant proteins into CEN.PK background Saccharomyces cerevisiae to obtain a Saccharomyces cerevisiae strain that can express recombinant proteins. This allows the performance of the engineered Saccharomyces cerevisiae strain resistant to oxidative stress in expressing exogenous proteins under oxidative stress to be determined by detecting the expression level of recombinant proteins.

[0012] The plasmid expressing the recombinant protein is preferably pAlphaAmyCPOT (PNAS2018,115,E11025-E11032).

[0013] The recombinant protein is preferably α-amylase.

[0014] The CEN.PK background is preferably strain MSBP003.

[0015] The overexpression is preferably achieved by homologous recombination to integrate the overexpressed gene into the chromosome.

[0016] The preferred site for chromosome integration is the X3 neutral site.

[0017] The knockout is preferably performed without scarring by homologous recombination or CRISPR / Cas9 gene editing technology.

[0018] Both the BIO5 and HMO1 genes are components of the Saccharomyces cerevisiae genome and can be obtained by PCR amplification using the Saccharomyces cerevisiae genome as a template.

[0019] The oxidative stress was induced by treating the Saccharomyces cerevisiae with a certain amount of either tunicamycin (Tm) or dithiothreitol (DTT), two stress inducers.

[0020] The method for constructing the above-mentioned engineered strain of *Saccharomyces cerevisiae* resistant to antioxidant stress includes the following steps: knocking out gene BIO5 and overexpressing gene HMO1; preferably, it includes the following steps:

[0021] (1) Using plasmid pROS13 as a template, the kanamycin resistance gene expression cassette (kanMX) containing the homologous arm of the BIO5 site was amplified using primer pair KBIO5P1 / KBIO5P2. The nucleic acid sequence of the kanamycin resistance gene expression cassette is shown in SEQ ID NO.1.

[0022] (2) Obtain the genomic DNA of the yeast starting strain;

[0023] (3) Using the genomic DNA from step (2) as a template, the HMO1 gene fragment was amplified using primer pair PHMO1P1 / PHMO1P2. The coding nucleic acid sequence of HMO1 is shown in SEQ ID NO.2.

[0024] (4) Construct the pROS10-X3 knockout plasmid. Using plasmid pROS10 as a template, the plasmid framework was amplified by primer pair pROS10FraP1 / pROS10FraP2; the gene fragment containing the 20bp target sequence gRNA at the X3 site was amplified by primer pair pROS10-N20-up / pROS10-N20-X3; the plasmid framework and the gene fragment were connected by Gibson assembly technology to obtain the pROS10-X3 knockout plasmid.

[0025] (5) Using plasmid p426GPD as a template, the plasmid framework was amplified by primer pair p426FraP1 / p426FraP2. The plasmid framework and the HMO1 gene fragment obtained in step (3) were connected by Gibson assembly technology to obtain p426-HMO1 plasmid.

[0026] (6) Using p426-HMO1 plasmid as template, the GPDp-HMO1-CYC1t gene repair fragment containing homologous arms was amplified using primer pair X3-up-GPD / X3-down-CYC1. Its nucleotide sequence is shown in SEQ ID NO.3.

[0027] (7) Transform the pROS10-X3 knockout plasmid obtained in step (4) and the GPDp-HMO1-CYC1t gene repair fragment obtained in step (6) into the starting strain to obtain strain R2;

[0028] (8) The kanamycin resistance gene expression cassette obtained in step (1) was transformed into competent cells of strain R2 to obtain an engineered strain of Saccharomyces cerevisiae resistant to oxidative stress.

[0029] Application of the above-mentioned engineered strains of Saccharomyces cerevisiae with antioxidant stress in oxidative stress environments.

[0030] Application of the above-mentioned engineered strains of Saccharomyces cerevisiae that resist oxidative stress in the production of recombinant proteins.

[0031] A method for testing the antioxidant stress performance of the above-mentioned engineered Saccharomyces cerevisiae strains includes the following steps:

[0032] 1) The above-mentioned engineered strains of Saccharomyces cerevisiae subjected to antioxidant stress were inoculated into fermentation media without Tm / DTT, containing 0.2 μg / mL and 0.4 μg / mL Tm, and containing 3 mM and 5 mM DTT, respectively, and the cell growth was detected.

[0033] 2) Centrifuge the fermented bacterial broth and collect the supernatant for determination of recombinant protein content;

[0034] 3) The antioxidant stress performance of the above-mentioned engineered Saccharomyces cerevisiae strains was obtained by analyzing cell growth and recombinant proteins.

[0035] The fermentation medium mentioned in step 1) is preferably SD-2×SCAA medium.

[0036] The preferred culture conditions described in step 1) are 28–32°C and 150–250 rpm for 90–100 h; more preferably, 30°C and 200 rpm for 96 h.

[0037] The preferred conditions for centrifuging the bacterial culture in step 2) are 10,000 to 13,000 × g for 0 to 2 min; more preferably, 12,000 × g for 1 min.

[0038] The content of recombinant protein described in step 2) was determined by using an α-amylase assay kit (K-CERA, Megazyme) to measure the enzyme activity of α-amylase, while Aspergillus oryzae α-amylase (Sigma-Aldrich) was used as a standard control.

[0039] The present invention has the following advantages and effects compared with the prior art:

[0040] This invention modifies yeast strains expressing recombinant proteins, successfully constructing an engineered strain with simultaneously enhanced oxidative stress resistance and recombinant protein production. This invention identified two effective targets, BIO5 and HMO1, and combined them to construct strain R3, which exhibits the greatest improvement in stress resistance. Under oxidative stress induced by 0.4 μg / mTtm and 5 mM DTT, strain R3 showed higher cell density and α-amylase production than the starting strain R0. Specifically, under DTT treatment, the cell density of strain R3 increased significantly, being twice that of the control strain; under Tm and DTT treatments, the recombinant protein production of strain R3 increased by 2.14-fold and 1.85-fold, respectively. The key targets we discovered may contribute to the design and construction of high-protein-secreting, stress-resistant strains, improving the efficiency of recombinant protein production using Saccharomyces cerevisiae in industry and bringing greater economic benefits. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of the recombinant plasmid; where (A) is p426-HMO1 and (B) is pROS10-X3.

[0042] Figure 2 This is an agarose gel electrophoresis image of strain R1, which was identified by PCR with BIO5 knocked out; lane M is the DNA molecular weight marker.

[0043] Figure 3 This is a gel electrophoresis image of PCR identification of the HMO1 gene successfully integrated into the R2 strain at the X3 site; lane M is the DNA molecular weight marker.

[0044] Figure 4 This is a gel electrophoresis image of PCR identification of the HMO1 gene successfully integrated into the R3 strain at the X3 site; lane M is the DNA molecular weight marker.

[0045] Figure 5 This is a data graph showing the results of fermentation of R1, R2, and R3 yeast engineered strains in Example 5 for 4 days in a medium without Tm / DTT; R0 (AMP003) is used as a control.

[0046] Figure 6 The data graphs are of the yeast engineered strains R1, R2, and R3 in Example 6 after fermentation for 4 days in culture media containing 0.2 μg / mLTm (A) and 3 mMDTT (B), respectively; R0 (AMP003) is used as a control.

[0047] Figure 7 This is a graph showing the data after 4 days of fermentation of the R3 yeast engineered strain in test tubes in Example 7 in media containing 0.4 μg / mLTm and 5 mM DTT, respectively; R0 serves as the control. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0049] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0050] Unless otherwise specified, experimental methods in the following examples are generally performed under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (Beijing: Science Press, 2017) and Yeast Genetics: A Laboratory Manual (Beijing: Science Press, 2016).

[0051] The CRISPR technology used in the following embodiments is described in the prior art (Nat Commun 2019, 10(1):1053).

[0052] The plasmid p426GPD used in the following examples has been disclosed in the literature “Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 1995, 156: 119-122.”; the plasmid pAlphaAmyCPOT has been disclosed in the literature “Engineering the protein secretory pathway of Saccharomyces cerevisiae enables improved protein production. PNAS 2018, 115: E11025-E11032”; and the plasmids pROS10 and pROS13 have been disclosed in the literature “CRISPR / Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. FEMS Yeast Research, 2015, 15, fov004” and are available from EUROSCARF.

[0053] To better understand the content of this invention, based on the background strain MSBP003 of Saccharomyces cerevisiae CEN.PK [Comprehensive analysis of signal peptides in Saccharomyces cerevisiae reveals features for efficient secretion. Advanced Science 10, 2203433 (2023)] (available from EUROSCARF), specific embodiments are further illustrated.

[0054] The culture media involved in the following examples are as follows:

[0055] LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, solvent is deionized water; solid medium is supplemented with 2% agar powder.

[0056] LB / AMP medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, solvent is deionized water; solid medium with 2% agar powder added; after sterilization, cool to about 40°C and add 100 μg / mL ampicillin (filtered for sterilization).

[0057] YPD medium: 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose (sterilized separately and then added), solvent is deionized water; solid medium is supplemented with 2% agar powder.

[0058] YPD+G418 solid medium: 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose (sterilized separately and then added), solvent is deionized water; 2% agar powder is added to the solid medium; after sterilization, cool to about 40°C and add 200 μg / mL LG418 (filtered for sterilization).

[0059] SC-Ura auxotrophic medium: 0.77 g / L CSM-Ura, 1.7 g / L YNB w / o AA & w / o (NH4)2SO4 (Yeast Nitrogen Base without Amino acids and without Ammonium sulphate), 5.0 g / L (NH4)2SO4, 20 g / L glucose (Note: glucose is sterilized separately), adjust pH to 5.5-6.0; add 2% agar powder to solid medium; solvent is deionized water.

[0060] SD-2×SCAA (addUra) medium: 13.6 g / L Na2HPO4·12H2O, 11.1 g / L NaH2PO4·2H2O, 1.7 g / L YNB (w / o AA, w / o ammonia sulfate), 5 g / L (NH4)2SO4, 40 mg / L LUra, 190 mg / L Arg, 400 mg / L Asp, 1260 mg / L Glu, 130 mg / L Gly, 140 mg / L His, 290 mg / L Ile, 400 mg / L Leu, 440 mg / L Lys, 108 mg / L Met, 200 mg / L Phe, 220 mg / L Thr, 40 mg / L Trp, 52 mg / L Tyr, 380 mg / L LVal, 20 g / L glucose (Note: glucose is sterilized separately), adjust pH to 6.0.

[0061] The methods involved in the following embodiments are as follows:

[0062] Plasmid construction:

[0063] (1) Gibson assembly method, the specific operation shall be carried out in accordance with the NEB Gibson Assembly Cloning kit (part number E2611, NEB) instruction manual;

[0064] (2) Transform 10 μL of the assembly system into 50 μL of E. coli DH5α competent cells, spread them on LB / AMP solid medium and culture overnight;

[0065] (3) Positive clones were obtained by screening, and plasmids were extracted after amplification and culture. The specific extraction process was carried out in accordance with the instructions of HiPure Plasmid Micro Kit (product number P1001-03, Magen).

[0066] Yeast strain transformation:

[0067] Unless otherwise specified, the lithium acetate conversion method shall be used. For specific operation procedures, please refer to the relevant standards and specifications.

[0068] Recombinant Saccharomyces cerevisiae strain OD 600nm Detection methods:

[0069] A single colony of *Saccharomyces cerevisiae* was inoculated into 2 mL of fermentation medium and cultured on a shaker at 30°C and 200 rpm. The fermentation broth was then diluted appropriately and its OD (exposure potential) was measured using a UV spectrophotometer. 600nm .

[0070] The primer sequences involved in the following examples are shown in Table 1:

[0071] Table 1: Primer sequences (5'-3')

[0072]

[0073] Example 1: Construction of starting strain R0

[0074] The plasmid pAlphaAmyCPOT was transformed into the CEN.PK background starting strain MSBP003 using the lithium acetate transformation method. The transformed strain was then plated onto a YPD solid culture base and cultured at 30°C for 3–4 days. Positive transformants were obtained, and the Saccharomyces cerevisiae strain AMP003 was obtained and named R0.

[0075] Example 2: Construction of p426-HMO1 plasmid

[0076] The structure of plasmid p426-HMO1 is as follows: Figure 1 As shown in (A), the specific construction process is as follows:

[0077] (1) Using primer pair P426FraP1 / P426FraP2, the plasmid framework was amplified using p426GPD plasmid as template; PCR amplification included routine denaturation, annealing and extension steps; the annealing temperature was 56℃, the extension temperature was 72℃ for 200s, and 30 cycles were performed.

[0078] (2) Using primer pair PHMO1P1 / PHMO1P2, the CDS fragment of the HMO1 gene with homologous arms was amplified using the genome of the lysed R0 strain as a template; the annealing temperature of PCR amplification was 56℃, the extension temperature was 72℃ for 25s, and 30 cycles were performed.

[0079] (3) Using Gibson assembly technology, the two fragments above were spliced ​​together and transformed into Escherichia coli DH5α. The resulting plasmid was named p426-HMO1.

[0080] Example 3: Construction of engineered strain R1 with BIO5 gene knocked out

[0081] (1) Using primer pair KBIO5P1 / KBIO5P2, the kanamycin resistance gene expression cassette (kanMX) containing the homologous arm of the BIO5 site was amplified using plasmid pROS13 as a template; PCR amplification included routine denaturation, annealing and extension steps; the annealing temperature was 56℃, the extension temperature was 72℃ for 44s, and 30 cycles were performed.

[0082] (2) The kanMX fragment was transformed into the starting strain R0 using the lithium acetate transformation method. The transformed strain was plated onto a YPD+G418 solid culture medium and incubated at 30℃ for 3–4 days. Single-colony PCR was performed using primers kanIP-up / KBIO5IP to verify the transformations and screen for positive transformants (results). Figure 2As shown in the figure, the obtained Saccharomyces cerevisiae strain was named R1.

[0083] Example 4: Construction of engineered strain R2 overexpressing the HMO1 gene

[0084] (1) Construction of pROS10-X3

[0085] Using plasmid pROS10 as a template, a plasmid framework with the selection marker URA3 was amplified using primer pair PSNR52 / PSNR53. PCR amplification was performed with an annealing temperature of 56℃, an extension at 72℃ for 108 s, and 30 cycles. Using plasmid pROS10 as a template, a fragment with a 20 bp target to the X3 site (gRNA sequence) was amplified using primer pair PX3N20-up / PX3N20-down. PCR amplification was performed with an annealing temperature of 56℃, an extension at 72℃ for 37 s, and 30 cycles. The two fragments were then spliced ​​together using Gibson assembly technology and transformed into *E. coli* DH5α. The resulting plasmid was named pROS10-X3 (structure shown below). Figure 1 (as shown in (B)).

[0086] (2) PCR amplification of HMO1 repair fragment

[0087] Using primers X3-up-GPD / X3-down-CYC1, the GPDp-HMO1-CYC1t gene fragment with the X3 site homologous arm was amplified using p426-HMO1 plasmid as a template. This fragment served as the repair fragment. The PCR amplification annealing temperature was 56℃, followed by an extension at 72℃ for 52s, and 30 cycles were performed.

[0088] (3) The pROS10-X3 plasmid and HMO1 repair fragment were transformed into strain R0 using the lithium acetate transformation method. The transformed strains were plated onto SC-Ura solid culture bases and incubated at 30℃ for 3–4 days. Single-colony PCR was performed using primers to verify the X3-up-IP / OEHMO1 colony, and positive transformants were screened (results). Figure 3 As shown in the figure, the obtained Saccharomyces cerevisiae strain was named R2.

[0089] Example 5: Construction of engineered Saccharomyces cerevisiae R3

[0090] Saccharomyces cerevisiae strain R2 was prepared as competent cells. The kanMX fragment was transformed into R2 competent cells using the lithium acetate transformation method. The transformed cells were plated on YPD+G418 solid culture base and incubated at 30℃ for 3–4 days. Single-colony PCR was performed using primers kanIP-up / KBIO5IP to verify the transformations and screen for positive transformants. (Results) Figure 4 As shown in the figure, a modified Saccharomyces cerevisiae strain was obtained and named R3.

[0091] Example 6: Fermentation test of engineered strains under no external pressure.

[0092] (1) The above-mentioned Saccharomyces cerevisiae strains R0 (control), R1, R2 and R3 were inoculated from the plate into 2 mL of SD-2×SCAA fermentation medium and cultured at 30℃ and 200 rpm for 96 h.

[0093] (2) After diluting the fermentation broth 30 times, measure the OD using a UV spectrophotometer. 600nm .

[0094] (3) After centrifuging 1 mL of fermentation broth, take the supernatant, dilute it appropriately, and then determine the yield of recombinant protein.

[0095] according to Figure 5 The results show that after 4 days of fermentation in a medium without Tm and DTT, the constructed strains R1, R2, and R3 grew normally. Furthermore, the recombinant protein secretion levels of these strains were significantly increased, reaching 35%, 59%, and 88%, respectively.

[0096] Example 7: Antioxidant stress test of engineered strains

[0097] (1) The above-mentioned Saccharomyces cerevisiae strains R0 (control), R1, R2 and R3 were inoculated from the plate into 2 mL of SD-2×SCAA+0.2μg / mL Tm and SD-2×SCAA+3mMDTT medium, respectively, and cultured at 30℃ and 200rpm for 96h.

[0098] (2) After diluting the fermentation broth 30 times, measure the OD using a UV spectrophotometer. 600nm .

[0099] (3) After centrifuging 1 mL of fermentation broth, take the supernatant, dilute it appropriately, and then measure the yield of recombinant protein.

[0100] The results of fermentation of the constructed strains R1, R2, and R3 in SD-2×SCAA + 0.2 μg / mL Tm medium for 4 days are as follows: Figure 6 As shown in (A), the results after 4 days of fermentation in SD-2×SCAA+3mMDTT medium are as follows. Figure 6 As shown in (B), knocking out the BIO5 gene, overexpressing the HMO1 gene, and the combination of both do not affect the growth of the strain (reflected in cell density). Under Tm oxidative stress, the R3 strain's ability to produce recombinant proteins is twice that of the control strain.

[0101] Example 8: Antioxidant stress test of engineered strains (higher dose Tm / DTT)

[0102] (1) The above-mentioned Saccharomyces cerevisiae strains R0 (control) and R3 were inoculated from the plates into 2 mL of SD-2×SCAA+0.4μg / mL Tm and SD-2×SCAA+5mMDTT medium, respectively, and cultured at 30℃ and 200rpm for 96h.

[0103] (2) After diluting the fermentation broth 30 times, measure the OD using a UV spectrophotometer. 600nm .

[0104] (3) After centrifuging 1 mL of fermentation broth, take the supernatant, dilute it appropriately, and then measure the yield of recombinant protein.

[0105] The results of fermenting the constructed R3 strain for 4 days in SD-2×SCAA+0.4μg / mL Tm and SD-2×SCAA+5mMDTT media are as follows: Figure 7 As shown, the R3 engineered strain exhibited better growth, with a more pronounced advantage under DTT treatment, showing a 2-fold increase in cell density compared to the control strain. Under higher doses of Tm oxidative stress, the R3 strain demonstrated 2.14 times the protein production capacity of the control strain.

[0106] The engineered strain R3 exhibits significantly enhanced resistance to oxidative stress and significantly improved recombinant protein production capacity. This stress-resistant strain has broad application prospects and reference value.

[0107] This invention provides an antioxidant-resistant Saccharomyces cerevisiae genetically engineered strain and its construction method. There are many methods and approaches to implement this technical solution. The above description is only a preferred embodiment of this invention. The implementation of this invention is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of this invention should be considered equivalent substitutions and are included within the protection scope of this invention.

Claims

1. An engineered strain of *Saccharomyces cerevisiae* resistant to oxidative stress, characterized in that... It has the following characteristics: genes BIO5 Knockout, gene HMO1 Overexpression.

2. The engineered strain of *Saccharomyces cerevisiae* resistant to antioxidant stress according to claim 1, characterized in that: The starting strain of the engineered Saccharomyces cerevisiae is CEN.PK background Saccharomyces cerevisiae.

3. The engineered strain of *Saccharomyces cerevisiae* with antioxidant stress according to claim 1, characterized in that: The plasmid expressing the recombinant protein was introduced into CEN.PK background Saccharomyces cerevisiae to obtain a Saccharomyces cerevisiae strain expressing the recombinant protein.

4. The engineered strain of *Saccharomyces cerevisiae* with antioxidant stress according to claim 3, characterized in that: The recombinant protein is α-amylase; The CEN.PK background Saccharomyces cerevisiae is strain MSBP003.

5. The engineered strain of *Saccharomyces cerevisiae* with antioxidant stress according to claim 1, characterized in that: The overexpression mentioned refers to the overexpression of genes through chromosomal integration via homologous recombination. The knockout is performed without scarring by homologous recombination or CRISPR / Cas9 gene editing technology.

6. The method for constructing the engineered strain of *Saccharomyces cerevisiae* with antioxidant stress according to any one of claims 1 to 5, characterized in that... The steps include: gene knockout BIO5 overexpression of genes HMO1 .

7. The construction method according to claim 6, characterized in that... Includes the following steps: (1) Using plasmid pROS13 as a template, primer pair KBIO5P1 / KBIO5P2 was used to amplify plasmids containing pROS13. BIO5 The kanamycin resistance gene expression cassette with a site homologous arm, the nucleic acid sequence of which is shown in SEQ ID NO.1; (2) Obtain the genomic DNA of the yeast starting strain; (3) Using the genomic DNA from step (2) as a template, amplify using primer pair PHMO1P1 / PHMO1P2. HMO1 Gene fragments, HMO1 The encoding nucleic acid sequence is shown in SEQ ID NO.2; (4) Construct the pROS10-X3 knockout plasmid. Using plasmid pROS10 as a template, the plasmid framework was amplified by primer pair pROS10FraP1 / pROS10FraP2; the gene fragment containing the 20bp target sequence gRNA at the X3 site was amplified by primer pair pROS10-N20-up / pROS10-N20-X3; the plasmid framework and the gene fragment were connected by Gibson assembly technology to obtain the pROS10-X3 knockout plasmid. (5) Using plasmid p426GPD as a template, the plasmid framework was amplified by primer pair p426FraP1 / p426FraP2. The plasmid framework and the plasmid obtained in step (3) were then assembled using Gibson assembly technology. HMO1 Gene fragments were ligated to obtain the p426-HMO1 plasmid; (6) Using p426-HMO1 plasmid as a template, the GPDp- containing homologous arms was amplified using primer pair X3-up-GPD / X3-down-CYC1. HMO1 - CYC1 The t gene repair fragment, the nucleotide sequence of which is shown in SEQ ID NO.3; (7) Combine the pROS10-X3 knockout plasmid obtained in step (4) with the GPDp- obtained in step (6). HMO1 - CYC1 The t gene repair fragment was transformed into the starting strain to obtain strain R2; (8) Transform the kanamycin resistance gene expression cassette obtained in step (1) into the competent cells of strain R2 to obtain an engineered strain of Saccharomyces cerevisiae resistant to oxidative stress.

8. The application of the engineered strain of Saccharomyces cerevisiae according to any one of claims 1 to 5 in an oxidative stress environment.

9. The use of the antioxidant-stress-resistant Saccharomyces cerevisiae engineered strain according to any one of claims 1 to 5 in the production of recombinant proteins.

10. A method for testing the antioxidant stress performance of an engineered strain of *Saccharomyces cerevisiae* according to any one of claims 1 to 5, characterized in that... Includes the following steps: 1) The engineered strain of Saccharomyces cerevisiae with antioxidant stress according to any one of claims 1 to 5 was inoculated into fermentation medium without Tm / DTT, containing 0.2 μg / mL and 0.4 μg / mL Tm concentrations, and containing 3 mM and 5 mM DTT concentrations, and the cell growth was detected. 2) Centrifuge the fermented bacterial broth and collect the supernatant for determination of recombinant protein content; 3) The antioxidant stress performance of the above-mentioned engineered Saccharomyces cerevisiae strains was obtained by analyzing cell growth and recombinant proteins.

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