Multi-dimensional synergistically regulated saccharomyces cerevisiae engineering bacterium for producing recombinant human serum albumin as well as construction method and application of saccharomyces cerevisiae engineering bacterium

Abstract

The invention discloses a multi-dimensional synergistically regulated saccharomyces cerevisiae engineering bacterium for producing recombinant human serum albumin as well as a construction method and application of the saccharomyces cerevisiae engineering bacterium. The invention systematically verifies the effects of independent knockout and combined knockout of genes of PEP1, PEP4, PRB1, PRC1, YPS1, YAP3, CPS1, PEA1 and the like on strain protein production in saccharomyces cerevisiae, and determines that the double deletion combination of PEP4 and PEP1 is an optimal modification strategy for resisting degradation and secretion enhancement of human serum albumin. Targeted transformation is further carried out on genes related to the secretion pathway of chassis cells, and organic combination of endoplasmic reticulum lipid expansion and form remodeling is achieved by knocking out OPI1 and overexpressing RTN2 at the same time. The multi-dimensional combination strategy generates a remarkable synergistic effect, and the secretion yield of the recombinant human serum albumin is remarkably improved compared with that of an original strain while the degradation impurity bands of the recombinant human serum albumin are reduced.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and microbial technology, and specifically relates to a multidimensional synergistically regulated engineered Saccharomyces cerevisiae strain that produces recombinant human serum albumin, its construction method, and its application. Background Technology

[0002] Human serum albumin (HSA) is the most abundant protein in human plasma, synthesized by the liver at a daily rate of approximately 10-15 grams. HSA plays a crucial role in various physiological processes, including maintaining plasma osmotic pressure and intravascular fluid balance; acting as a carrier to transport hormones, fatty acids, and other small molecules; regulating capillary permeability; participating in anti-inflammatory and antioxidant responses; and regulating coagulation function. Furthermore, HSA can neutralize free radicals, reducing their oxidative damage to the body. Clinically, the demand for HSA is extremely high, and it is widely used in the treatment of diseases such as blood loss, shock, burns, and cirrhosis. Currently, its main source is extraction from healthy human plasma, but this method faces three major bottlenecks: first, a shortage of blood supply, making it difficult to meet the continuously growing clinical demand; second, high production costs, limiting its widespread application; and third, the potential risk of viral contamination (such as HIV, hepatitis B virus, and hepatitis C virus), threatening medication safety. In addition, plasma extraction also faces ethical restrictions and difficulties in quality control. Therefore, developing a method to produce recombinant human serum albumin efficiently and safely will greatly promote its application in the fields of medicine and biotechnology, not only meeting current needs but also better addressing future challenges.

[0003] Saccharomyces cerevisiae, as a recognized eukaryotic model organism, has been widely used for the secretory expression of recombinant proteins. Its genetic background is clear, it is easy to manipulate genetically, and it can be fermented on a large scale. It possesses a complete eukaryotic protein folding and post-translational modification system, which can mimic the protein processing process in human cells. It can secrete target proteins extracellularly, reducing downstream purification costs. Furthermore, Saccharomyces cerevisiae has been designated a Generally Recognized as Safe (GRAS) species by the FDA, indicating high safety of its expression system and the absence of endotoxin contamination risk. It is considered one of the promising expression hosts for recombinant human serum albumin.

[0004] However, current Saccharomyces cerevisiae expression systems commonly suffer from protein degradation during the production of recombinant human serum albumin, hindering its industrial application. Studies show that yeast regulates its protease system according to environmental changes during growth to synthesize and degrade different proteins and organelles. Under stress conditions such as late-stage fermentation or high-density culture, cell quality control mechanisms are activated, and vacuolar proteases may be released into the cytoplasm or accidentally secreted extracellularly, causing hydrolysis of exogenous proteins. This not only significantly reduces the yield of the target protein but also significantly increases purification difficulty, thus hindering the large-scale production of recombinant human serum albumin. Therefore, there is an urgent need to construct an engineered Saccharomyces cerevisiae strain that can reduce protein degradation while improving secretion efficiency to achieve efficient and stable expression of recombinant human serum albumin and meet the needs of industrial production. Summary of the Invention

[0005] To address the common technical problems of severe product degradation and limited secretion efficiency in the production of recombinant human serum albumin using existing Saccharomyces cerevisiae expression systems, this invention aims to provide a degradation-resistant and high-yielding Saccharomyces cerevisiae strain for recombinant human serum albumin production, along with its construction method and applications. This invention employs a systematic modification strategy combining inhibition of product degradation and expansion of the secretion pathway. The specific technical solution is as follows: First, this invention tests various single protease gene deletion strains. The results show that the PEP4 gene deletion strain is most effective in reducing recombinant human serum albumin degradation, suggesting that the vacuolar degradation pathway is one of the key factors limiting recombinant human serum albumin yield. To further solve the problems of residual degradation and secretion bottlenecks, this invention performs secondary knockout screening on a series of protease and vacuolar sorting-related genes, including PEP1, PRB1, PRC1, YPS1, YAP3, CPS1, and PEA1, in the PEP4 gene deletion strain. The experimental results creatively revealed that, unlike the conventionally chosen double-deletion combination of PEP4 and PRB1, the double-deletion combination of PEP4 and PEP1 (encoding the vacuole protein sorting receptor Vps10p) genes exhibited the best synergistic effect in resisting degradation and increasing recombinant human serum albumin production, significantly outperforming other double-deletion combinations. Based on the aforementioned optimal double-deletion strain, further modifications were made to key nodes affecting protein secretion, including endoplasmic reticulum morphology regulators (RTN1, RTN2, YOP1) and lipid synthesis regulators (OPI1). The results showed that knocking out OPI1 (a negative regulator of phospholipid synthesis) and overexpressing RTN2 (an endoplasmic reticulum morphology protein) both effectively increased recombinant human serum albumin production. Finally, this invention combines effective modification strategies, further knocking out OPI1 and simultaneously overexpressing RTN2 on the basis of the PEP4 and PEP1 double-deletion strain to obtain the final engineered strain. This combined modification exhibits synergistic effects in three dimensions: reducing product degradation, increasing endoplasmic reticulum membrane capacity, and optimizing endoplasmic reticulum morphology, resulting in a significant increase in the production of recombinant human serum albumin.

[0006] The primary objective of this invention is to provide a multidimensionally synergistically regulated engineered Saccharomyces cerevisiae strain that produces recombinant human serum albumin.

[0007] Another objective of this invention is to provide a method for constructing the above-mentioned engineered Saccharomyces cerevisiae strain that produces recombinant human serum albumin under multidimensional synergistic regulation.

[0008] Another object of the present invention is to provide the application of the above-mentioned engineered Saccharomyces cerevisiae in the production of recombinant human serum albumin and / or in increasing the secretory expression level of recombinant human serum albumin.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A multidimensional synergistically regulated Saccharomyces cerevisiae producing recombinant human serum albumin, having at least one of the characteristics (2) to (12) and characteristic (1); preferably having at least one of the characteristics (2) to (8) and characteristic (1), or having at least one of the characteristics (9) to (12) and characteristic (1) and characteristic (2);

[0011] (1) Knock out the PEP4 gene and overexpress human serum albumin;

[0012] (2) Knock out the PEP1 gene;

[0013] (3) Knock out the PRB1 gene;

[0014] (4) Knock out the PRC1 gene;

[0015] (5) Knock out the YPS1 gene;

[0016] (6) Knock out the YAP3 gene;

[0017] (7) Knock out the CPS1 gene;

[0018] (8) Knock out the PEA1 gene;

[0019] (9) Knock out the OPI1 gene;

[0020] (10) Knock out the RTN1 gene;

[0021] (11) Knock out the YOP1 gene;

[0022] (12) Overexpression of RTN2p.

[0023] The preferred Saccharomyces cerevisiae strain for producing recombinant human serum albumin under the above-mentioned multidimensional synergistic regulation is any one of the following strains:

[0024] Strain T1: PEP4 gene knocked out, PEP1 gene knocked out;

[0025] Strain T8: Knockout of PEP4 gene, knockout of PEP1 gene, knockout of OPI1 gene;

[0026] Strain T9: Knockout of PEP4 gene, knockout of PEP1 gene, knockout of RTN1 gene;

[0027] Strain T10: PEP4 gene knocked out, PEP1 gene knocked out, YOPI1 gene knocked out;

[0028] Strain G1: PEP4 gene knockout, PEP1 gene knockout, RTN2p overexpression;

[0029] Strain T12: PEP4 gene knockout, PEP1 gene knockout, OPI1 gene knockout, RTN2p overexpression.

[0030] The substrate strain of the engineered Saccharomyces cerevisiae is Saccharomyces cerevisiae; preferably, it is a Saccharomyces cerevisiae strain with a CEN.PK background; more preferably, it is the Saccharomyces cerevisiae AH30 strain.

[0031] The amino acid sequence of the human serum albumin is shown in SEQ ID NO.1.

[0032] The nucleotide sequence of the PEP4 gene is shown in SEQ ID NO.4.

[0033] The nucleotide sequence of the PEP1 gene is shown in SEQ ID NO.5.

[0034] The nucleotide sequence of the PRB1 gene is shown in SEQ ID NO.6.

[0035] The nucleotide sequence of the PRC1 gene is shown in SEQ ID NO.7.

[0036] The nucleotide sequence of the YPS1 gene is shown in SEQ ID NO.8.

[0037] The nucleotide sequence of the YAP3 gene is shown in SEQ ID NO9.

[0038] The nucleotide sequence of the CPS1 gene is shown in SEQ ID NO.10.

[0039] The nucleotide sequence of the PEA1 gene is shown in SEQ ID NO.11.

[0040] The nucleotide sequence of the OPI1 gene is shown in SEQ ID NO.12.

[0041] The nucleotide sequence of the RTN1 gene is shown in SEQ ID NO.13.

[0042] The nucleotide sequence of the YOP1 gene is shown in SEQ ID NO.14.

[0043] The nucleotide sequence of the RTN2 gene is shown in SEQ ID NO.15.

[0044] The overexpression of RTN2p is preferably achieved by transforming a plasmid expressing the RTN2 gene into a Saccharomyces cerevisiae strain.

[0045] The knockout is preferably performed without scarring using CRISPR / Cas9 gene editing technology.

[0046] The engineered Saccharomyces cerevisiae contains an expression plasmid for human serum albumin.

[0047] The method for constructing the above-mentioned engineered Saccharomyces cerevisiae strain producing recombinant human serum albumin through multidimensional synergistic regulation includes the following steps:

[0048] 1) The human serum albumin expression vector was transformed into Saccharomyces cerevisiae strain AH30 to obtain strain D0;

[0049] 2) Knock out the PEP4 gene in the genome of strain D0 to obtain strain D1;

[0050] 3) Knock out the PEP1 gene in the genome of strain D0 to obtain strain D2;

[0051] 4) Knock out the PRB1 gene in the genome of strain D0 to obtain strain D3;

[0052] 5) Knock out the PRC1 gene in the genome of strain D0 to obtain strain D4;

[0053] 6) Knock out the YPS1 gene in the genome of strain D0 to obtain strain D5;

[0054] 7) Knock out the YAP3 gene in the genome of strain D0 to obtain strain D6;

[0055] 8) Knock out the CPS1 gene in the genome of strain D0 to obtain strain D7;

[0056] 9) Knock out the PEA1 gene in the genome of strain D0 to obtain strain D8;

[0057] 10) Perform one of the following operations on the yeast engineered strain D1 obtained in step 2):

[0058] S1. Knock out the PEP1 gene in the genome of strain D1 to obtain strain T1;

[0059] S2. Knock out the PRB1 gene in the genome of strain D1 to obtain strain T2;

[0060] S3. Knock out the PRC1 gene in the genome of strain D1 to obtain strain T3;

[0061] S4. Knock out the YPS1 gene in the genome of strain D1 to obtain strain T4;

[0062] S5. Knock out the YAP3 gene in the genome of strain D1 to obtain strain T5;

[0063] S6. Knock out the CPS1 gene in the genome of strain D1 to obtain strain T6;

[0064] S7. Knock out the PEA1 gene in the genome of strain D1 to obtain strain T7;

[0065] 11) Perform one or more of the following operations on the yeast strain T1 obtained in step 10):

[0066] S1. Knock out the OPI1 gene in the genome of strain T1 to obtain strain T8;

[0067] S2. Knock out the RTN1 gene in the genome of strain T1 to obtain strain T9;

[0068] S3. Knock out the YOP1 gene in the genome of strain T1 to obtain strain T10;

[0069] S4. Transform the RTN2 expression vector into T1 to obtain strain G1;

[0070] 12) The RTN2 expression vector was transformed into strain T8 to obtain strain T12.

[0071] The human serum albumin expression vector mentioned in step 1) is preferably an expression vector containing nucleic acid with the sequence shown in SEQ ID NO.2; more preferably, it is plasmid pCP-AHSA.

[0072] In step 11) S4, the RTN2 expression vector is preferably an expression vector containing nucleic acid with the sequence shown in SEQ ID NO.15; more preferably, it is plasmid p416-RTN2.

[0073] The RTN2 expression vector mentioned in step 12) is preferably an expression vector containing nucleic acid with the sequence shown in SEQ ID NO.15; more preferably, it is plasmid p416-RTN2.

[0074] The application of the above-mentioned engineered Saccharomyces cerevisiae in the production of recombinant human serum albumin and / or in improving the secretory expression level of recombinant human serum albumin; preferably includes the following steps: fermenting the above-mentioned engineered Saccharomyces cerevisiae in a fermentation medium, centrifuging to remove the cells after fermentation, and obtaining a supernatant containing recombinant human serum albumin.

[0075] The fermentation medium is preferably SD-2×SCAA+Ura medium or SD-2×SCAA-Ura medium.

[0076] The fermentation culture conditions are preferably 20–40 °C and 100–300 rpm for 84–108 h; more preferably 30 °C and 215 rpm for 96 h.

[0077] The invention has the following advantages and effects compared to the prior art:

[0078] This invention constructs a multidimensionally synergistically regulated Saccharomyces cerevisiae engineered strain that produces recombinant human serum albumin, effectively reducing the degradation during the secretion and expression process of recombinant human serum albumin and significantly increasing the yield of recombinant human serum albumin.

[0079] Unlike traditional methods that modify protease genes individually, this invention systematically screens eight different proteases and related genes, innovatively proposing a synergistic modification strategy that combines degradation pathway blocking and secretion competition resolution to improve degradation. On one hand, knocking out the PEP4 gene blocks the cascade activation pathway of vacuolar hydrolases, directly eliminating the specific hydrolytic effect of protein A on human serum albumin. On the other hand, knocking out the PEP1 gene blocks the directional sorting of endogenous hydrolases into vacuoles, reducing the risk of intracellular accumulation and accidental release of hydrolases, and avoiding competition and congestion with the human serum albumin secretion pathway. The synergistic effect of these two methods is superior to all other combinations, effectively solving the degradation problem unique to human serum albumin during fermentation, significantly increasing the yield of recombinant human serum albumin in the fermentation supernatant, and virtually eliminating degradation contaminants. Based on the optimal anti-degradation strain, this invention further reveals the key limiting role of endoplasmic reticulum (ER) morphology and lipid synthesis in the secretion of recombinant human serum albumin. A synergistic breakthrough in yield was achieved by jointly knocking out the negative regulator of phospholipid synthesis (OPI1) and simultaneously overexpressing the network structure protein (RTN2). This multi-level, multi-target systematic optimization strategy resulted in a significant increase in the recombinant human serum albumin yield of the final engineered strain.

[0080] The yeast genetically engineered strain of this invention significantly enhances the strain's ability to secrete complete recombinant human serum albumin. The reduction in degradation fragments lowers the complexity and cost of downstream separation and purification, solving the pain points of difficult purification and low yield in the industrial production of recombinant human serum albumin. It shows great potential in increasing the yield of proteins with high economic value and has broad application prospects. Attached Figure Description

[0081] Figure 1 This is PCR verification of the knockout of the genome target in the engineered strain. Lane M is the Marker GeneRuler 1kb DNA Ladder; Lane 2 is the PEP4 knockout strain D1 of this invention; Lane 4 is the PEP1 knockout strain T1 of this invention; Lanes 1 and 3 are the starting strain D0 without PEP4 and PEP1 knockout; Lane 5 is the OPI1 knockout strain T8 of this invention; and Lane 6 is the RTN2p overexpressing strain T12 of this invention.

[0082] Figure 2This is an SDS-PAGE image of the fermentation supernatant of a single knockout protease gene strain; in the image, lane M is the Marker GeneRuler 1kb DNA Ladder, lanes 1 and 2 are strain D1, lanes 3 and 4 are strain D2, lanes 5 and 6 are strain D3, lanes 7 and 8 are strain D4, lanes 9 and 10 are strain D5, lanes 11 and 12 are strain D6, lanes 13 and 14 are strain D7, lanes 15 and 16 are strain D8, and lanes 17-18 are the starting strain D0.

[0083] Figure 3 This is a graph showing the relative quantitative results of human serum albumin products from the fermentation supernatant of a double knockout protease gene strain compared to the starting strain D0.

[0084] Figure 4 These are SDS-PAGE images of the fermentation supernatants of strains D0, D1, and T1; lane M is the MarkerGeneRuler 1kb DNA Ladder, lane 1 is the starting strain D0, lane 2 is the single gene deletion strain D1 with PEP4 knocked out, and lane 3 is the double gene deletion strain T1 with both PEP4 and PEP1 knocked out.

[0085] Figure 5 This is a graph showing the ELISA results of human serum albumin concentration in the fermentation supernatant of strains D0, D1, and T1.

[0086] Figure 6 This is a graph showing the relative quantitative results of human serum albumin products from the supernatant of a genetically engineered strain with secretion pathway-related genes compared to the double-gene deletion strain T1.

[0087] Figure 7 This is a Western Blot result of the fermentation supernatant of the multidimensionally combined engineered strain; in the figure, lane M is the Marker GeneRuler 1kb DNA Ladder, lanes 1-2 are strain T12 of strain T8 that overexpresses RTN2, and lanes 3-4 are strain T11 of strain T1 that overexpresses the empty vector plasmid p416-GPD.

[0088] Figure 8 This is a graph showing the relative quantitative results of human serum albumin products from the fermentation supernatant of the multidimensionally engineered strain T12 compared to strain T11, which overexpresses the empty vector plasmid. Detailed Implementation

[0089] 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.

[0090] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0091] Experimental methods in the following examples that do not specify specific conditions 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).

[0092] The CRISPR technology used in the following embodiments is described in the prior art (Nat. Commun. 2019, 10:1053).

[0093] The plasmids pROS10 and p416-GPD used in the following examples are disclosed in the literature “Improved protein production in yeast using cell engineering with genes related to a key factor in the unfolded protein response. Metab Eng. 2023, 77:152-161.” (see supplementary materials for that literature for details). The plasmid pCP-AHSA is disclosed in the literature “Efficient protein production by yeast requires global tuning of metabolism. Nat. Commun. 2017, 8, 1131.” (see supplementary materials for that literature for details).

[0094] To better understand the content of this invention, specific embodiments are further described based on the background strain AH30 of Saccharomyces cerevisiae CEN.PK [disclosed in the literature "Optimization of Protein Folding for Improved Secretion of Human Serum Albumin Fusion Proteins in Saccharomyces cerevisiae. J AgricFood Chem. 2023, 29, 71(47):18414-18423.", see the supplementary materials of that literature for details] and the Saccharomyces cerevisiae strain CEN.PK 530-1D [available from EUROSCARF].

[0095] The culture media and reagents used in the following examples are as follows:

[0096] LB / AMP 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 by weight. After sterilization, the above are cooled to about 40 ℃, and ampicillin is added to a final concentration of 100 µg / mL (filtered for sterilization).

[0097] 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% (w / v) agar powder.

[0098] SD-Ura auxotrophic medium: 0.77 g / L CSM-Ura, 1.7 g / L YNB w / o aa and (NH4)2SO4, 5.0 g / L (NH4)2SO4, 20 g / L glucose (Note: glucose is sterilized separately), adjust the pH to 5.5-6.0, and use deionized water as the solvent; add 2% (w / v) agar powder to the solid medium.

[0099] Fermentation medium SD-2×SCAA+Ura: 1.7 g / L YNB (w / o AA, w / o ammonia sulfate), 5.0 g / L (NH4)2SO4, 13.6 g / L Na2HPO4·12H2O, 9.7 g / L NaH2PO4·2H2O, adjusted pH to 6.0, autoclaved, then glucose was added to a final concentration of 20 g / L, along with SCAA solution (190 mg / L Arg, 108 mg / L Met, 52 mg / L Tyr, 290 mg / L Ile, 440 mg / L Lys, 200 mg / L Phe, 1260 mg / L Glu, 400 mg / L Lasp, 380 mg / L Val, 220 mg / L Thr, 130 mg / L Gly, 400 mg / L Leu, 40 mg / L Trp, 140 mg / L His, 40 mg / L... Ura), the solvent is deionized water. Note: Glucose is sterilized separately and sterilized by SCAA filtration.

[0100] Fermentation medium SD-2×SCAA-Ura: 1.7 g / L YNB (w / o AA, w / o ammonia sulfate), 5.0 g / L (NH4)2SO4, 13.6 g / L Na2HPO4·12H2O, 9.7 g / L NaH2PO4·2H2O, adjusted pH to 6.0, autoclaved, then glucose to a final concentration of 20 g / L and SCAA solution (190 mg / L Arg, 108 mg / L Met, 52 mg / L Tyr, 290 mg / L Ile, 440 mg / L Lys, 200 mg / L Phe, 1260 mg / L Glu, 400 mg / L Lasp, 380 mg / L Val, 220 mg / L Thr, 130 mg / L Gly, 400 mg / L Leu, 40 mg / L Trp, 140 mg / L His), in deionized water. Note: Glucose is sterilized separately and then filtered and sterilized using SCAA.

[0101] Electrophoresis buffer: Tris 60.6 g / L, MOPS 104.6 g / L, SDS 10 g / L, EDTA 3 g / L, solvent: deionized water. Store at room temperature.

[0102] Transfer buffer: Glycine 15.11 g / L, Tris 3.03 g / L, methanol 200 mL / L, solvent: deionized water. The prepared solution was filtered for sterilization, degassed by sonication, and stored at room temperature.

[0103] The methods involved in the following embodiments are as follows:

[0104] Plasmid construction:

[0105] (1) Gibson assembly method; follow the instructions of NEB Gibson Assembly Cloning kit (part number E2611, NEB) for specific operations;

[0106] (2) Transform 5 µL of Gibson assembly system into 50 µL of E. coli DH5α competent cells, spread them on LB / AMP solid medium and culture overnight;

[0107] (3) Screening to obtain positive clones, amplification culture and plasmid extraction, the specific extraction process is carried out in accordance with the instructions of HiPure Plasmid Micro Kit (product number P1001-03, Magen).

[0108] Preparation of competent yeast strains:

[0109] (1) Dispense 100 mL of the bacterial cells obtained from the expansion culture into 50 mL centrifuge tubes, set the speed to 4200 rpm, centrifuge at room temperature for 5 min, discard the supernatant and harvest the bacterial cells;

[0110] (2) Wash the bacterial cells with 20 mL of sterile water, set the speed to 4200 rpm, centrifuge at room temperature for 5 min and then discard the supernatant;

[0111] (3) Resuspend the bacterial cells in 0.5 mL of sterile water, combine and transfer to a sterile centrifuge tube, centrifuge again at 8000 rpm for 1 min, remove the supernatant, add 1 mL (1 / 100 of the total volume of the culture system) of FCC (5% v / v glycerol, 10% v / v dimethyl sulfoxide) solution to resuspend the bacterial cells, and store in an ultra-low temperature freezer at -80 ℃.

[0112] Yeast strain transformation:

[0113] Unless otherwise specified, the lithium acetate conversion method shall be used. For specific operation procedures, please refer to the relevant standards and specifications.

[0114] Yeast strain OD 600nm Detection methods:

[0115] A single colony of *Saccharomyces cerevisiae* was inoculated into 2.5 mL of fermentation medium (SD-2×SCAA+Ura) and cultured on a shaker at 30 ℃ and 215 rpm. The fermentation broth was diluted appropriately and the OD was measured using a UV spectrophotometer. 600nm .

[0116] Methods for relative and precise quantification of human serum albumin levels:

[0117] (1) Relative quantification: The main band of human serum albumin (approximately 66.5 kDa) in SDS-PAGE fractions was analyzed by grayscale scanning using ImageJ software.

[0118] (2) Accurate quantification: Follow the instructions of the Human Albumin ELISA Kit (catalog number KE00076, Proteintech).

[0119] The primer sequences involved in the following examples are shown in Tables 1 and 2:

[0120] Table 1: Primer sequences

[0121]

[0122] Table 2: Primer sequences

[0123]

[0124] Example 1 Systematic screening of high-yield recombinant human serum albumin-resistant yeast strains

[0125] The background strain for the yeast genetically engineered strain in this embodiment is Saccharomyces cerevisiae AH30.

[0126] 1.1 Expression of recombinant human serum albumin by Saccharomyces cerevisiae strain

[0127] The Saccharomyces cerevisiae strain AH30 was prepared into competent cells. The plasmid pCP-AHSA (expressing recombinant human serum albumin, the amino acid sequence of which is shown in SEQ ID NO.1, and the sequence encoding the nucleic acid is shown in SEQ ID NO.2) was transformed into competent cells of strain AH30 using the lithium acetate conversion method. The transformed cells were then plated on YPD culture media and cultured at 30 °C for 3–4 days. The resulting strain was named D0.

[0128] 1.2 Knockout of the PEP4 gene

[0129] The PEP4 knockout process is as follows:

[0130] (1) Construction of plasmid pROS10-PEP4

[0131] The nucleotide sequence of PEP4 is shown in SEQ ID NO.4.

[0132] Using plasmid pROS10 as a template, a 1458 bp gRNA fragment targeting the PEP4 gene was amplified using primer pair Q-PEP4-F / URA3-R. PCR amplification was performed with denaturation at 95℃ for 15 s, annealing at 56℃ for 15 s, and extension at 72℃ for 60 s, for 30 cycles (the amplification conditions for the gRNA fragment below are the same as here).

[0133] Using plasmid pROS10 as a template, a 3307 bp plasmid framework fragment pROS10-k with the selection marker URA3 was amplified by primer pair URA3-F / dqPROS10-R (nucleotide sequence shown in SEQ ID NO.3); PCR amplification was performed at 95℃ for denaturation for 15 s, 56℃ for annealing for 15 s, and 72℃ for extension for 120 s, for 30 cycles.

[0134] Using Gibson assembly technology, a 20 bp gRNA targeting the PEP4 gene was spliced ​​with pROS10-k, and the resulting plasmid was named pROS10-PEP4.

[0135] (2) PCR amplification of PEP4 repair fragment

[0136] Using the CEN.PK 530-1D genome as a template, the PEP4-targeted repair fragment with 50 bp homologous arms upstream and downstream of the PEP4 gene was obtained by PCR amplification and gel recovery using primers dPEP4-1 / dPEP4-2. The PCR amplification was performed at 95℃ for 15 s denaturation, 56℃ for 15 s annealing, and 72℃ for 15 s extension for 30 cycles (the amplification conditions for the repair fragments below are the same as here).

[0137] (3) Prepare competent cells of yeast strain D0. Transform plasmid pROS10-PEP4 and PEP4 targeted repair fragment into D0 competent cells simultaneously by lithium acetate transformation. Spread the cells on SD-Ura solid culture base and culture at 30 ℃ for 3-4 days. Use primers JPEP4-1 / JPEP4-2 to perform single colony PCR verification and screen for positive transformants.

[0138] (4) The positive transformants were streaked onto 5-FOA solid medium and incubated at 30 °C for 3-4 days to remove the target plasmid. The resulting single colonies were then spotted onto SD-Ura and YPD solid medium for verification. Only single colonies growing on YPD medium were identified as yeast strains with the pROS10-PEP4 plasmid removed. This strain was named Saccharomyces cerevisiae D1.

[0139] 1.3 Knockout of the PEP1 gene

[0140] The PEP1 knockout process is as follows:

[0141] (1) Construction of plasmid pROS10-PEP1

[0142] The nucleotide sequence of PEP1 is shown in SEQ ID NO.5.

[0143] Using plasmid pROS10 as a template, a 1458 bp gRNA fragment targeting the PEP1 gene was amplified by primer pair Q-PEP1-F / URA3-R.

[0144] Using Gibson assembly technology, a 20 bp gRNA targeting the PEP1 gene was spliced ​​with the pROS10-k plasmid framework, and the resulting plasmid was named pROS10-PEP1.

[0145] (2) PCR amplification of PEP1 repair fragment

[0146] Using the CEN.PK 530-1D genome as a template, the PEP1 targeted repair fragment with 50 bp homologous arms upstream and downstream of the PEP1 gene was obtained by PCR amplification and gel recovery using primers dPEP1-1 / dPEP1-2.

[0147] (3) The plasmid pROS10-PEP1 and the PEP1 targeted repair fragment were simultaneously transformed into D0 competent cells by lithium acetate transformation. The cells were then plated on SD-Ura solid culture base and cultured at 30 °C for 3-4 days. Single colony PCR was performed using primers JPEP1-1 / JPEP1-2 to verify the transformations and positive transformants were obtained.

[0148] (4) The positive transformants were streaked onto 5-FOA solid medium and incubated at 30 °C for 3-4 days to remove the target plasmid. The resulting single colonies were then spotted onto SD-Ura and YPD solid medium for verification. Only single colonies growing on YPD medium were identified as yeast strains with the pROS10-PEP1 plasmid removed. This strain was named Saccharomyces cerevisiae D2.

[0149] 1.4 Knockout of PRB1 gene

[0150] The PRB1 knockout process is as follows:

[0151] (1) Construction of plasmid pROS10-PRB1

[0152] The nucleotide sequence of PRB1 is shown in SEQ ID NO.6.

[0153] Using plasmid pROS10 as a template, a 1458 bp gRNA fragment targeting the PRB1 gene was amplified by primer pair Q-PRB1-F / URA3-R.

[0154] Using Gibson assembly technology, a 20 bp gRNA targeting the PRB1 gene was spliced ​​together with the pROS10-k plasmid framework, and the resulting plasmid was named pROS10-PRB1.

[0155] (2) PCR amplification of PRB1 repair fragment

[0156] Using the CEN.PK 530-1D genome as a template, the PRB1 targeted repair fragment with 50 bp homologous arms upstream and downstream of the PRB1 gene was obtained by PCR amplification and gel recovery using primers dPRB1-1 / dPRB1-2.

[0157] (3) The plasmid pROS10-PRB1 and the PRB1 targeted repair fragment were simultaneously transformed into D0 competent cells by lithium acetate transformation. The cells were then plated on SD-Ura solid culture base and cultured at 30 °C for 3-4 days. Single colony PCR was performed using primers JPRB1-1 / JPRB1-2 to verify the transformations and positive transformants were obtained.

[0158] (4) The positive transformants were streaked onto 5-FOA solid medium and incubated at 30 °C for 3-4 days to remove the target plasmid. The resulting single colonies were then spotted onto SD-Ura and YPD solid medium for verification. Only single colonies growing on YPD medium were identified as yeast strains with the pROS10-PRB1 plasmid removed. This strain was named Saccharomyces cerevisiae D3.

[0159] 1.5 Knockout of the PRC1 gene

[0160] The PRC1 knockout process is as follows:

[0161] (1) Construction of plasmid pROS10-PRC1

[0162] The nucleotide sequence of PRC1 is shown in SEQ ID NO.7.

[0163] Using plasmid pROS10 as a template, a 1458 bp gRNA fragment targeting the PRC1 gene was amplified by primer pair Q-PRC1-F / URA3-R.

[0164] Using Gibson assembly technology, a 20 bp gRNA targeting the PRC1 gene was spliced ​​together with the pROS10-k plasmid framework, and the resulting plasmid was named pROS10-PRC1.

[0165] (2) PCR amplification of PRC1 repair fragment

[0166] Using the CEN.PK 530-1D genome as a template, the PRC1 targeted repair fragment with 50 bp homologous arms upstream and downstream of the PRC1 gene was obtained by PCR amplification and gel recovery using primers dPRC1-1 / dPRC1-2.

[0167] (3) The plasmid pROS10-PRC1 and the PRC1 targeted repair fragment were simultaneously transformed into D0 competent cells by lithium acetate transformation. The cells were then plated on SD-Ura solid culture base and cultured at 30 °C for 3-4 days. Single colony PCR was performed using primers JPRC1-1 / JPRC1-2 to verify the transformations and positive transformants were obtained.

[0168] (4) The positive transformants were streaked onto 5-FOA solid medium and incubated at 30 °C for 3-4 days to remove the target plasmid. The resulting single colonies were then spotted onto SD-Ura and YPD solid medium for verification. Only single colonies growing on YPD medium were identified as yeast strains with the pROS10-PRC1 plasmid removed. This strain was named Saccharomyces cerevisiae D4.

[0169] 1.6 Knockout of the YPS1 gene

[0170] The process of knocking out YPS1 is as follows:

[0171] (1) Construction of plasmid pROS10-YPS1

[0172] The nucleotide sequence of YPS1 is shown in SEQ ID NO.8.

[0173] Using plasmid pROS10 as a template, a 1458 bp gRNA fragment targeting the YPS1 gene was amplified by primer pair Q-YPS1-F / URA3-R.

[0174] Using Gibson assembly technology, a 20 bp gRNA targeting the YPS1 gene was spliced ​​with the pROS10-k plasmid framework, and the resulting plasmid was named pROS10-YPS1.

[0175] (2) PCR amplification of YPS1 repair fragment

[0176] Using the CEN.PK 530-1D genome as a template, the YPS1 targeted repair fragment with 50 bp homologous arms upstream and downstream of the YPS1 gene was obtained by PCR amplification and gel recovery using primers dYPS1-1 / dYPS1-2.

[0177] (3) The plasmid pROS10-YPS1 and the YPS1 targeted repair fragment were simultaneously transformed into D0 competent cells by lithium acetate transformation. The cells were then plated on SD-Ura solid culture base and cultured at 30 °C for 3-4 days. Single colony PCR was performed using primers JYPS1-1 / JYPS1-2 to verify the transformations and positive transformants were obtained.

[0178] (4) The positive transformants were streaked onto 5-FOA solid medium and incubated at 30 °C for 3-4 days to remove the target plasmid. The resulting single colonies were then spotted onto SD-Ura and YPD solid medium for verification. Only single colonies growing on YPD medium were identified as yeast strains with the pROS10-YPS1 plasmid removed. This strain was named Saccharomyces cerevisiae D5.

[0179] 1.7 Knockout of the YAP3 gene

[0180] The process of knocking out YAP3 is as follows:

[0181] (1) Construction of plasmid pROS10-YAP3

[0182] The nucleotide sequence of YAP3 is shown in SEQ ID NO.9.

[0183] Using plasmid pROS10 as a template, a 1458 bp gRNA fragment targeting the YAP3 gene was amplified by primer pair Q-YAP3-F / URA3-R.

[0184] Using Gibson assembly technology, a 20 bp gRNA targeting the YAP3 gene was spliced ​​with the pROS10-k plasmid framework, and the resulting plasmid was named pROS10-YAP3.

[0185] (2) PCR amplification of YPS3 repair fragment

[0186] Using the CEN.PK 530-1D genome as a template, the YAP3 targeted repair fragment with 50 bp homologous arms upstream and downstream of the YAP3 gene was obtained by PCR amplification and gel recovery using primers dYAP3-1 / dYAP3-2.

[0187] (3) The plasmid pROS10-YAP3 and the YAP3 targeted repair fragment were simultaneously transformed into D0 competent cells by lithium acetate transformation. The cells were then plated on SD-Ura solid culture base and cultured at 30 °C for 3-4 days. Single colony PCR was performed using primers JYAP3-1 / JYAP3-2 to verify the transformations and positive transformants were obtained.

[0188] (4) The positive transformants were streaked onto 5-FOA solid medium and incubated at 30 °C for 3-4 days to remove the target plasmid. The resulting single colonies were then spotted onto SD-Ura and YPD solid medium for verification. Only single colonies growing on YPD medium were identified as yeast strains with the pROS10-YAP3 plasmid removed. This strain was named Saccharomyces cerevisiae D6.

[0189] 1.8 CPS1 gene knockout

[0190] The CPS1 knockout process is as follows:

[0191] (1) Construction of plasmid pROS10-CPS1

[0192] The nucleotide sequence of CPS1 is shown in SEQ ID NO.10.

[0193] Using plasmid pROS10 as a template, a 1458 bp gRNA fragment targeting the CPS1 gene was amplified by primer pair Q-CPS1-F / URA3-R.

[0194] Using Gibson assembly technology, a 20 bp gRNA targeting the CPS1 gene was spliced ​​together with the pROS10-k plasmid framework, and the resulting plasmid was named pROS10-CPS1.

[0195] (2) PCR amplification of CPS1 repair fragment

[0196] Using the CEN.PK 530-1D genome as a template, the CPS1 targeted repair fragment with 50 bp homologous arms upstream and downstream of the CPS1 gene was obtained by PCR amplification and gel recovery using primers dCPS1-1 / dCPS1-2.

[0197] (3) The plasmid pROS10-CPS1 and the CPS1 targeted repair fragment were simultaneously transformed into D0 competent cells by lithium acetate transformation. The cells were then plated on SD-Ura solid culture base and cultured at 30 °C for 3-4 days. Single colony PCR was performed using primers JCPS1-1 / JCPS1-2 to verify the transformations and positive transformants were obtained.

[0198] (4) The positive transformants were streaked onto 5-FOA solid medium and incubated at 30 °C for 3-4 days to remove the target plasmid. The resulting single colonies were then spotted onto SD-Ura and YPD solid medium for verification. Only single colonies growing on YPD medium were identified as yeast strains with the pROS10-CPS1 plasmid removed. This strain was named Saccharomyces cerevisiae D7.

[0199] 1.9 Knockout of PEA1 gene

[0200] The PEA1 knockout process is as follows:

[0201] (1) Construction of plasmid pROS10-PEA1

[0202] The nucleotide sequence of PEA1 is shown in SEQ ID NO.11.

[0203] Using plasmid pROS10 as a template, a 1458 bp gRNA fragment targeting the PEA1 gene was amplified by primer pair Q-PEA1-F / URA3-R.

[0204] Using Gibson assembly technology, a 20 bp gRNA targeting the PEA1 gene was spliced ​​together with the pROS10-k plasmid framework, and the resulting plasmid was named pROS10-PEA1.

[0205] (2) PCR amplification of PEA1 repair fragment

[0206] Using the CEN.PK 530-1D genome as a template, the PEA1 targeted repair fragment with 50 bp homologous arms upstream and downstream of the PEA1 gene was obtained by PCR amplification and gel recovery using primers dPEA1-1 / dPEA1-2.

[0207] (3) The plasmid pROS10-PEA1 and the PEA1 targeted repair fragment were simultaneously transformed into D0 competent cells by lithium acetate transformation. The cells were then plated on SD-Ura solid culture base and cultured at 30 °C for 3-4 days. Single colony PCR was performed using primers JPEA1-1 / JPEA1-2 to verify the transformations and positive transformants were obtained.

[0208] (4) The positive transformants were streaked onto 5-FOA solid medium and incubated at 30 °C for 3-4 days to remove the target plasmid. The resulting single colonies were then spotted onto SD-Ura and YPD solid medium for verification. Only single colonies growing on YPD medium were identified as yeast strains with the pROS10-PEA1 plasmid removed. This strain was named Saccharomyces cerevisiae D8.

[0209] 1.10 Knock out the PEP4 gene while simultaneously knocking out the PEP1 gene.

[0210] (1) Yeast strain D1 was prepared into competent cells. The plasmid pROS10-PEP1 and the PEP1 targeted repair fragment were simultaneously transformed into D1 competent cells by lithium acetate transformation. The cells were plated on SD-Ura solid culture base and cultured at 30 °C for 3-4 days. Single colony PCR was performed using primers JPEP1-1 / JPEP1-2 to verify the results and screen for positive transformants.

[0211] (2) Positive transformants were streaked onto 5-FOA solid medium and cultured at 30 °C for 3-4 days to remove the target plasmid. Single colonies were spotted onto SD-Ura and YPD solid medium for verification. Only single colonies growing on YPD medium were identified as yeast strains with the pROS10-PEP1 plasmid removed. This strain was named Saccharomyces cerevisiae T1.

[0212] 1.11 Knock out the PEP4 gene while simultaneously knocking out the PRB1 gene.

[0213] (1) Yeast strain D1 was prepared into competent cells. The plasmid pROS10-PRB1 and the PRB1 targeted repair fragment were simultaneously transformed into D1 competent cells by lithium acetate transformation. The cells were plated on SD-Ura solid culture base and cultured at 30 °C for 3-4 days. Single colony PCR was performed using primers JPRB1-1 / JPRB1-2 to verify the results and screen for positive transformants.

[0214] (2) Positive transformants were streaked onto 5-FOA solid medium and cultured at 30 °C for 3-4 days to remove the target plasmid. Single colonies were spotted onto SD-Ura and YPD solid medium for verification. Single colonies growing only on YPD medium were yeast strains with the pROS10-PRB1 plasmid removed. This strain was named Saccharomyces cerevisiae T2.

[0215] 1.12 Knock out the PEP4 gene while simultaneously knocking out the PRC1 gene.

[0216] (1) Yeast strain D1 was prepared into competent cells. The plasmid pROS10-PRC1 and the PRC1 targeted repair fragment were simultaneously transformed into D1 competent cells by lithium acetate transformation. The cells were plated on SD-Ura solid culture base and cultured at 30 °C for 3-4 days. Single colony PCR was performed using primers JPRC1-1 / JPRC1-2 to verify the results and screen for positive transformants.

[0217] (2) Positive transformants were streaked onto 5-FOA solid medium and cultured at 30 °C for 3-4 days to remove the target plasmid. Single colonies were spotted onto SD-Ura and YPD solid medium for verification. Only single colonies growing on YPD medium were identified as yeast strains with the pROS10-PRC1 plasmid removed. This strain was named Saccharomyces cerevisiae T3.

[0218] 1.13 Knock out the PEP4 gene while simultaneously knocking out the YPS1 gene.

[0219] (1) Yeast strain D1 was prepared into competent cells. The plasmid pROS10-YPS1 and the YPS1 targeted repair fragment were simultaneously transformed into D1 competent cells by lithium acetate transformation. The cells were plated on SD-Ura solid culture base and cultured at 30 ℃ for 3-4 days. Single colony PCR was performed using primers JYPS1-1 / JYPS1-2 to verify and screen for positive transformants.

[0220] (2) Positive transformants were streaked onto 5-FOA solid medium and incubated at 30 °C for 3-4 days to remove the target plasmid. Single colonies were spotted onto SD-Ura and YPD solid medium for verification. Single colonies growing only on YPD medium were yeast strains with the pROS10-YPS1 plasmid removed. This strain was named Saccharomyces cerevisiae T4.

[0221] 1.14 Knock out the PEP4 gene while simultaneously knocking out the YAP3 gene.

[0222] (1) Yeast strain D1 was prepared into competent cells. The plasmid pROS10-YAP3 and the YAP3 targeted repair fragment were simultaneously transformed into D1 competent cells by lithium acetate transformation. The cells were plated on SD-Ura solid culture base and cultured at 30 °C for 3-4 days. Single colony PCR was performed using primers JYAP3-1 / JYAP3-2 to verify the results and screen for positive transformants.

[0223] (2) Positive transformants were streaked onto 5-FOA solid medium and incubated at 30 °C for 3-4 days to remove the target plasmid. Single colonies were spotted onto SD-Ura and YPD solid medium for verification. Only single colonies growing on YPD medium were identified as yeast strains with the pROS10-YAP3 plasmid removed. This strain was named Saccharomyces cerevisiae T5.

[0224] 1.15 Knock out the PEP4 gene while simultaneously knocking out the CPS1 gene.

[0225] (1) Yeast strain D1 was prepared into competent cells. The plasmid pROS10-CPS1 and the CPS1 targeted repair fragment were simultaneously transformed into D1 competent cells by lithium acetate transformation. The cells were plated on SD-Ura solid culture base and cultured at 30 °C for 3-4 days. Single colony PCR was performed using primers JCPS1-1 / JCPS1-2 to verify the results and screen for positive transformants.

[0226] (2) Positive transformants were streaked onto 5-FOA solid medium and cultured at 30 °C for 3-4 days to remove the target plasmid. Single colonies were spotted onto SD-Ura and YPD solid medium for verification. Only single colonies growing on YPD medium were identified as yeast strains with the pROS10-CPS1 plasmid removed. This strain was named Saccharomyces cerevisiae T6.

[0227] 1.16 Knock out the PEP4 gene while simultaneously knocking out the PEA1 gene.

[0228] (1) Yeast strain D1 was prepared into competent cells. The plasmid pROS10-PEA1 and the PEA1 targeted repair fragment were simultaneously transformed into D1 competent cells by lithium acetate transformation. The cells were plated on SD-Ura solid culture base and cultured at 30 °C for 3-4 days. Single colony PCR was performed using primers JPEA1-1 / JPEA1-2 to verify the results and screen for positive transformants.

[0229] (2) Positive transformants were streaked onto 5-FOA solid medium and cultured at 30 °C for 3-4 days to remove the target plasmid. Single colonies were spotted onto SD-Ura and YPD solid medium for verification. Only single colonies growing on YPD medium were identified as yeast strains with the pROS10-PEA1 plasmid removed. This strain was named Saccharomyces cerevisiae T7.

[0230] PCR validation results of key strains D1 and T1 are as follows Figure 1 As shown in Example 4, the relevant strains were fermented and human serum albumin was expressed. The results showed that the Saccharomyces cerevisiae strain T1 had the best protein expression effect and was selected as the chassis for subsequent modification.

[0231] Example 2: Effects of Single-Factor Modification of Endoplasmic Reticulum and Lipid Synthesis Pathway

[0232] 2.1 Knock out the PEP4 and PEP1 genes, and simultaneously knock out the OPI1 gene.

[0233] (1) Construction of plasmid pROS10-OPI1

[0234] The nucleotide sequence of OPI1 is shown in SEQ ID NO.12.

[0235] Using plasmid pROS10 as a template, a 1458 bp gRNA fragment targeting the OPI1 gene was amplified by primer pair Q-OPI1-F / URA3-R.

[0236] Using Gibson assembly technology, a 20 bp gRNA targeting the OPI1 gene was spliced ​​with the pROS10-k plasmid framework, and the resulting plasmid was named pROS10-OPI1.

[0237] (2) PCR amplification of OPI1 repair fragment

[0238] Using the CEN.PK 530-1D genome as a template, the OPI1 targeted repair fragment with 50 bp homologous arms upstream and downstream of the OPI1 gene was obtained by PCR amplification and gel recovery using primers dOPI1-1 / dOPI1-2.

[0239] (3) The plasmid pROS10-OPI1 and the OPI1 targeted repair fragment were simultaneously transformed into T1 competent cells by lithium acetate transformation. The cells were then plated on SD-Ura solid culture base and cultured at 30 °C for 3-4 days. Single colony PCR was performed using primers JOPI1-1 / JOPI1-2 to verify the transformations and positive transformants were obtained.

[0240] (4) The positive transformants were streaked onto 5-FOA solid medium and incubated at 30 °C for 3-4 days to remove the target plasmid. The resulting single colonies were then spotted onto SD-Ura and YPD solid medium for verification. Only single colonies growing on YPD medium were identified as yeast strains with the pROS10-OPI1 plasmid removed. This strain was named Saccharomyces cerevisiae T8.

[0241] 2.2 Knock out the PEP4 and PEP1 genes while simultaneously knocking out the RTN1 gene.

[0242] (1) Construction of plasmid pROS10-RTN1

[0243] The nucleotide sequence of RTN1 is shown in SEQ ID NO.13.

[0244] Using plasmid pROS10 as a template, a 1458 bp gRNA fragment targeting the RTN1 gene was amplified by primer pair Q-RTN1-F / URA3-R.

[0245] Using Gibson assembly technology, a 20 bp gRNA targeting the RTN1 gene was spliced ​​with the pROS10-k plasmid framework, and the resulting plasmid was named pROS10-RTN1.

[0246] (2) PCR amplification of RTN1 repair fragment

[0247] Using the CEN.PK 530-1D genome as a template, the RTN1 targeted repair fragment with 50 bp homologous arms upstream and downstream of the RTN1 gene was obtained by PCR amplification and gel recovery using primers dRTN1-1 / dRTN1-2.

[0248] (3) The plasmid pROS10-RTN1 and the RTN1 targeted repair fragment were simultaneously transformed into T1 competent cells by lithium acetate transformation. The cells were then plated on SD-Ura solid culture base and cultured at 30 °C for 3-4 days. Single colony PCR was performed using primers JRTN1-1 / JRTN1-2 to verify the transformations and positive transformants were obtained.

[0249] (4) The positive transformants were streaked onto 5-FOA solid medium and incubated at 30 °C for 3-4 days to remove the target plasmid. The resulting single colonies were then spotted onto SD-Ura and YPD solid medium for verification. Only single colonies growing on YPD medium were identified as yeast strains with the pROS10-RTN1 plasmid removed. This strain was named Saccharomyces cerevisiae T9.

[0250] 2.3 Knock out the YOP1 gene while simultaneously knocking out the PEP4 and PEP1 genes.

[0251] (1) Construction of plasmid pROS10-YOP1

[0252] The nucleotide sequence of YOP1 is shown in SEQ ID NO.14.

[0253] Using plasmid pROS10 as a template, a 1458 bp gRNA fragment targeting the YOP1 gene was amplified by primer pair Q-YOP1-F / URA3-R.

[0254] Using Gibson assembly technology, a 20 bp gRNA targeting the YOP1 gene was spliced ​​together with the pROS10-k plasmid framework, and the resulting plasmid was named pROS10-YOP1.

[0255] (2) PCR amplification of YOP1 repair fragment

[0256] Using the CEN.PK 530-1D genome as a template, the YOP1 targeted repair fragment with 50 bp homologous arms upstream and downstream of the YOP1 gene was obtained by PCR amplification and gel recovery using primers dYOP1-1 / dYOP1-2.

[0257] (3) The plasmid pROS10-YOP1 and YOP1 targeted repair fragment were simultaneously transformed into T1 competent cells by lithium acetate transformation. The cells were then plated on SD-Ura solid culture base and cultured at 30 °C for 3-4 days. Single colony PCR was performed using primers JYOP1-1 / JYOP1-2 to verify the transformations and positive transformants were obtained.

[0258] (4) The positive transformants were streaked onto 5-FOA solid medium and incubated at 30 °C for 3-4 days to remove the target plasmid. The resulting single colonies were then spotted onto SD-Ura and YPD solid medium for verification. Only single colonies growing on YPD medium were identified as yeast strains with the pROS10-YOP1 plasmid removed. This strain was named Saccharomyces cerevisiae T10.

[0259] 2.4 While knocking out PEP4 and PEP1 genes, overexpressing the RTN2 gene.

[0260] (1) Construction of plasmid p416-RTN2

[0261] The nucleotide sequence of RTN2 is shown in SEQ ID NO.15.

[0262] Using the CEN.PK 530-1D genome as a template, a 1239bp RTN2 gene fragment was amplified by primer pair RTN2-F / RTN2-R.

[0263] Using plasmid p416-GPD as a template, a 5717 bp plasmid framework fragment p416GPD-k (nucleotide sequence shown in SEQ ID NO.16) with the selection marker URA3 was amplified by primer pair p416-F / p416-R. PCR amplification consisted of denaturation at 95 ℃ for 15 s, annealing at 56 ℃ for 15 s, extension at 72 ℃ for 180 s, and 30 cycles.

[0264] Using Gibson assembly technology, the DNA fragment of the RTN2 gene was spliced ​​with the p416GPD-k plasmid framework, and the resulting plasmid was named p416-RTN2.

[0265] (2) The plasmid p416-RTN2 was transformed into T1 competent cells by lithium acetate transformation method, and then plated on SD-Ura solid culture base and cultured at 30 ℃ for 3-4 days. Single colony PCR was performed using primers p416-1 / p416-2 to verify the transformation. Positive transformants were screened and named Saccharomyces cerevisiae G1.

[0266] 2.5 Simultaneously, the empty vector p416-GPD was introduced while knocking out the PEP4 and PEP1 genes, serving as a control for the overexpression target strain.

[0267] (1) The plasmid p416-GPD was transformed into T1 competent cells by lithium acetate transformation method, and then plated on SD-Ura solid culture base and cultured at 30 ℃ for 3-4 days. Single colony PCR was performed using primers p416-1 / p416-2 to verify the transformation. Positive transformants were screened and named Saccharomyces cerevisiae T11 as a control for the overexpression strain.

[0268] PCR validation results of the key strain T8 are as follows Figure 1 As shown.

[0269] Example 3: Verification of the synergistic effect of multidimensional combination strategies

[0270] 3.1 While knocking out PEP4, PEP1, and OPI1 genes, the RTN2 gene was overexpressed.

[0271] (1) Construction of plasmid p416-RTN2

[0272] The nucleotide sequence of RTN2 is shown in SEQ ID NO.15.

[0273] Using the CEN.PK 530-1D genome as a template, a 1239bp RTN2 gene fragment was amplified by primer pair RTN2-F / RTN2-R.

[0274] Using Gibson assembly technology, the DNA fragment of the RTN2 gene was spliced ​​with the p416GPD-k plasmid framework, and the resulting plasmid was named p416-RTN2.

[0275] (2) Yeast strain T8 was prepared into competent cells. The plasmid p416-RTN2 was transformed into T8 competent cells using the lithium acetate transformation method. The cells were then plated on SD-Ura solid culture media and cultured at 30 ℃ for 3-4 days. Single-colony PCR verification was performed using primers p416-1 / p416-2. Positive transformants were screened and named *Saccharomyces cerevisiae* T12. The PCR verification results are as follows: Figure 1 As shown.

[0276] Example 4: Fermentation production of recombinant human serum albumin by engineered strains

[0277] (1) The above-mentioned Saccharomyces cerevisiae strains were inoculated from plates into 2.5 mL of SD-2×SCAA+Ura fermentation medium. Strains containing p416-RTN2 or p416GPD plasmids were fermented in SD-2×SCAA-Ura medium. The fermentation broth was prepared by culturing at 30 ℃ and 215 rpm for 96 h.

[0278] (2) Dilute the fermentation broth 50 times and measure the OD using a UV spectrophotometer. 600nm The fermentation broth was centrifuged at 8000 rpm for 2 min, and the supernatant was collected for SDS-PAGE or Western blotting analysis. The grayscale scan analysis of the human serum albumin master band (approximately 66.5 kDa) was performed using ImageJ software, and the relative expression level was calculated using the corresponding control strains as a baseline. Simultaneously, key strains (D0, D1, T1) were selected for quantitative determination using a human serum albumin-specific ELISA kit.

[0279] (3) Effect of single protease knockout on recombinant human serum albumin expression, such as Figure 2 As shown, compared with the starting strain D0, the strain D1 with the PEP4 gene knocked out had a significantly lighter degradation band at 45 kDa and the highest relative secretion yield. Strains with other protease genes knocked out (such as PRB1, YPS1, etc.) showed little improvement in degradation bands or a decrease in yield. These results indicate that PEP4 is the main factor leading to the degradation of recombinant human serum albumin. The PEP4 gene encodes vacuolar aspartic protease, which is a key initiating enzyme for the maturation and activation of various hydrolases in the vacuoles (such as protease B, carboxypeptidase Y, etc.). Knocking out PEP4 renders the main proteases in the vacuolars in an inactive prozymogen state, thereby fundamentally blocking the main pathway of human serum albumin degradation. Therefore, strain D1 was determined to be the optimal base strain for subsequent multi-gene combination modification.

[0280] (4) The screening results of secondary knockout in strain D1 are as follows: Figure 3As shown in the figure. The results indicated that, compared with strain D1, strains T1 and T6 produced increased recombinant human serum albumin, with strain T1 exhibiting the highest relative yield. Other double-gene deletion strains did not show an increase in recombinant human serum albumin production. The PEP1 (also known as VPS10) gene encodes a type I transmembrane receptor, primarily responsible for sorting and transporting various progenitors of hydrolases, such as carboxypeptidase Y (CPY), from the Golgi apparatus to the vacuole. Unlike directly knocking out protease genes (such as PRB1), knocking out PEP1 not only blocks the entry of residual hydrolases into the vacuole, but more importantly, it blocks the sorting and transport of a large number of endogenous vacuole proteins. This mechanism reduces the load on the Golgi apparatus and secretory pathway, decreasing competition for secretory resources from endogenous proteins, thereby freeing up cellular resources originally used for transporting vacuole proteins for the secretion of human serum albumin.

[0281] (5) To further confirm the performance of the key strains and verify the accuracy of the results, SDS-PAGE analysis and ELISA quantification were performed on the starting strain D0, the best single-gene deletion strain D1, and the best double-gene deletion strain T1. The SDS-PAGE results are as follows: Figure 4 As shown in the analysis, strain D0 exhibited a full-length band of recombinant human serum albumin at 66.5 kDa, but a dark band was observed at 45 kDa. Compared to the starting strain D0, the knockout of the PEP4 gene significantly lightened the specific degradation band, indicating that PEP4 knockout blocked the main degradation pathway. Dual knockout of both PEP4 and PEP1 genes further increased the width and color depth of the main band of recombinant human serum albumin, indicating increased yield. The concentration of recombinant human serum albumin in the fermentation supernatant was determined using a human serum albumin-specific ELISA kit. The results show that ( Figure 5 The recombinant human serum albumin yields of strains D0, D1, and T1 were 132.2 mg / L, 206.0 mg / L, and 252.8 mg / L, respectively. Compared to the starting strain D0, the yields of D1 and T1 increased by 56% and 91%, respectively. Considering both the degradation inhibition effect and the yield increase, the knockout of PEP4 and PEP1 was determined to be the optimal dual-gene knockout combination. PEP4 deletion reduces protease activity, while PEP1 deletion not only synergistically inhibits protease activity but may also reduce the load on the cellular secretory system by blocking the vacuole protein sorting pathway. The combination of the two achieves a dual effect of anti-degradation and secretion promotion, thereby further increasing the recombinant human serum albumin yield. Therefore, strain T1 was selected as the chassis for further optimization.

[0282] (6) To further explore the production potential of the T1 chassis, this invention investigated the influence of endoplasmic reticulum morphology and lipid synthesis-related genes. The results are as follows: Figure 6As shown, strain T8, with the OPI1 gene knocked out, exhibited the highest recombinant human serum albumin yield, a 23% increase compared to T1. Furthermore, strain G1, overexpressing the RTN2 gene, also showed a significant increase in recombinant human serum albumin yield, an 18% increase compared to T11 (strain T1 overexpressing an empty vector plasmid). The OPI1 gene encodes a transcriptional regulator involved in phospholipid metabolism. In this invention, the engineered strain obtained by deleting the OPI1 gene showed a significant increase in recombinant human serum albumin secretion yield. This result indicates that genetic modification of phospholipid metabolism regulators can effectively improve the production performance of secreted proteins in the system described in this invention. Its promoting effect is not based on a simple enhancement of a single metabolic pathway, but rather exhibits a clear overall improvement in secretory capacity. The RTN2 gene encodes an endoplasmic reticulum structure-related protein. This invention further found that overexpression of the RTN2 gene also resulted in a significant increase in recombinant human serum albumin yield in the aforementioned chassis strains. It is noteworthy that this enhancement effect cannot be directly predicted through linear inference from the general functions of endoplasmic reticulum structural proteins, indicating that the regulation of RTN2 expression levels has a specific and non-obvious promoting effect in the secretory protein production system of this invention. In summary, these results demonstrate that through combined screening and targeted regulation of membrane lipid metabolism regulators and endoplasmic reticulum structure-related factors, this invention has discovered a class of effective genetic modification targets capable of significantly enhancing the secretion efficiency of recombinant human serum albumin. In contrast, under the same experimental conditions, knockout modification of RTN1 or YOP1 did not produce the same degree of promotion, further illustrating that the roles of different endoplasmic reticulum-related factors in secretory protein production are significantly different and irreplaceable.

[0283] (7) Based on the above screening results, the present invention further constructed a combined modified strain. The results are as follows: Figure 7 and Figure 8 As shown, under the T1 background, strain T12, which simultaneously knocks out OPI1 and overexpresses RTN2, exhibits the highest relative yield of recombinant human serum albumin, increasing by 238% compared to T11 (the control strain obtained by introducing the empty vector p416-GPD into strain T1). Particularly noteworthy is that the yield increase of T12 is significantly greater than that of strain T8, which only knocks out OPI1, and strain G1, which only overexpresses RTN2. Furthermore, this is not a simple additive effect of knocking out OPI1 alone or overexpressing RTN2 alone, indicating a functional synergistic relationship between the two in the secretory protein production system described in this invention. Their synergistic effect is difficult to predict directly through linear inference before modification.

[0284] In summary, this invention successfully constructed a *Saccharomyces cerevisiae* strain that is resistant to degradation and efficiently secretes recombinant human serum albumin through a systematic screening and step-by-step modification strategy. Based on the experimental data from the above embodiments, the following conclusions can be drawn:

[0285] I. The strain with dual knockout of PEP4 and PEP1 was established as the optimal anti-degradation and secretion chassis.

[0286] Through systematic screening in Examples 1 and 2, this invention discovered that further knocking out the PEP1 gene, which encodes the vacuole protein sorting receptor, on the basis of PEP4 gene deletion, further increased the yield of recombinant human serum albumin and significantly reduced the degradation bands of the target protein. This effect was superior to the knockout of other protease genes such as PRB1 and CPS1. The dual knockout strategy of PEP4 and PEP1 not only optimized the anti-degradation of recombinant human serum albumin by inhibiting protease activity, but also achieved a breakthrough increase in recombinant human serum albumin yield by eliminating the secretion competition between endogenous and exogenous proteins. The yield was 91% higher than the starting strain, superior to other double-gene deletion strains, highlighting the synergistic innovative value of this modification strategy.

[0287] II. The key role of endoplasmic reticulum volume expansion and morphological remodeling in recombinant human serum albumin secretion was revealed.

[0288] Example 2 of this invention further systematically explored the endoplasmic reticulum and secretory pathway of the optimal basic strain T1. Based on the T1 chassis, this invention further confirmed that the endoplasmic reticulum is the rate-limiting step in the secretory expression of recombinant human serum albumin. This invention found that knocking out OPI1, knocking out RTN1, or overexpressing RTN2 genes can effectively increase the yield of recombinant human serum albumin. This indicates that increasing the membrane capacity of the endoplasmic reticulum is crucial for the correct folding and effective secretion of human serum albumin. This invention also found that knocking out YOP1 actually led to a decrease in the yield of recombinant human serum albumin, indicating that blindly destroying endoplasmic reticulum structural proteins will impair its secretory function. The above results indicate that the modification of endoplasmic reticulum-related genes is not a simple routine attempt, and its results are difficult to predict. The effective targets (OPI1, RTN1, RTN2) screened by this invention have outstanding substantial characteristics.

[0289] Third, it achieved synergistic effects from multi-dimensional transformation strategies, with significant results.

[0290] The combined engineered strain constructed in Example 3 of this invention significantly increased the yield of recombinant human serum albumin based on multiple genetic modifications. Specifically, PEP4 deletion effectively reduced the degradation of recombinant human serum albumin, and the double deletion combination of PEP4 and PEP1 showed superior effects compared to other double gene deletion combinations in reducing degradation during secretion and increasing yield. Furthermore, the introduction of OPI1 deletion and RTN2 overexpression further increased the recombinant human serum albumin yield of the resulting combined strain, exceeding that of any single or double-modified strain. These results demonstrate that through synergistic regulation of multiple targets, the expression and secretion levels of recombinant human serum albumin can be effectively enhanced in the system described in this invention.

[0291] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A multidimensionally synergistically regulated engineered Saccharomyces cerevisiae strain producing recombinant human serum albumin, characterized in that: The engineered brewer's yeast has at least one of the characteristics (2) to (12) and characteristic (1); (1) Knock out the PEP4 gene and overexpress human serum albumin; (2) Knock out the PEP1 gene; (3) Knock out the PRB1 gene; (4) Knock out the PRC1 gene; (5) Knock out the YPS1 gene; (6) Knock out the YAP3 gene; (7) Knock out the CPS1 gene; (8) Knock out the PEA1 gene; (9) Knock out the OPI1 gene; (10) Knock out the RTN1 gene; (11) Knock out the YOP1 gene; (12) Overexpression of RTN2p.

2. The engineered brewer's yeast strain according to claim 1, characterized in that: The engineered brewer's yeast has at least one of the characteristics (2) to (8) and characteristic (1), or has at least one of the characteristics (9) to (12) and characteristics (1) and characteristic (2).

3. The engineered brewer's yeast strain according to claim 1, characterized in that: The *Saccharomyces cerevisiae* strain that produces recombinant human serum albumin under multidimensional synergistic regulation is any one of the following strains: Strain T1: PEP4 gene knocked out, PEP1 gene knocked out; Strain T8: Knockout of PEP4 gene, knockout of PEP1 gene, knockout of OPI1 gene; Strain T9: Knockout of PEP4 gene, knockout of PEP1 gene, knockout of RTN1 gene; Strain T10: PEP4 gene knocked out, PEP1 gene knocked out, YOPI1 gene knocked out; Strain G1: PEP4 gene knockout, PEP1 gene knockout, RTN2p overexpression; Strain T12: PEP4 gene knockout, PEP1 gene knockout, OPI1 gene knockout, RTN2p overexpression.

4. The engineered brewing yeast according to any one of claims 1 to 3, characterized in that: The chassis strain of the engineered Saccharomyces cerevisiae is a Saccharomyces cerevisiae strain with CEN.PK background.

5. The engineered brewing yeast according to any one of claims 1 to 3, characterized in that: The amino acid sequence of the human serum albumin is shown in SEQ ID NO.1; The nucleotide sequence of the PEP4 gene is shown in SEQ ID NO.4; The nucleotide sequence of the PEP1 gene is shown in SEQ ID NO.5; The nucleotide sequence of the PRB1 gene is shown in SEQ ID NO. 6; The nucleotide sequence of the PRC1 gene is shown in SEQ ID NO.7; The nucleotide sequence of the YPS1 gene is shown in SEQ ID NO8; The nucleotide sequence of the YAP3 gene is shown in SEQ ID NO.9; The nucleotide sequence of the CPS1 gene is shown in SEQ ID NO.10; The nucleotide sequence of the PEA1 gene is shown in SEQ ID NO.11; The nucleotide sequence of the OPI1 gene is shown in SEQ ID NO.12; The nucleotide sequence of the RTN1 gene is shown in SEQ ID NO.13; The nucleotide sequence of the YOP1 gene is shown in SEQ ID NO.14; The nucleotide sequence of the RTN2 gene is shown in SEQ ID NO.

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6. The method for constructing the engineered Saccharomyces cerevisiae producing recombinant human serum albumin under multidimensional synergistic regulation as described in any one of claims 1 to 5, characterized in that... Includes the following steps: 1) The human serum albumin expression vector was transformed into Saccharomyces cerevisiae strain AH30 to obtain strain D0; 2) Knock out the PEP4 gene in the genome of strain D0 to obtain strain D1; 3) Knock out the PEP1 gene in the genome of strain D0 to obtain strain D2; 4) Knock out the PRB1 gene in the genome of strain D0 to obtain strain D3; 5) Knock out the PRC1 gene in the genome of strain D0 to obtain strain D4; 6) Knock out the YPS1 gene in the genome of strain D0 to obtain strain D5; 7) Knock out the YAP3 gene in the genome of strain D0 to obtain strain D6; 8) Knock out the CPS1 gene in the genome of strain D0 to obtain strain D7; 9) Knock out the PEA1 gene in the genome of strain D0 to obtain strain D8; 10) Perform one of the following operations on the yeast engineered strain D1 obtained in step 2): S1. Knock out the PEP1 gene in the genome of strain D1 to obtain strain T1; S2. Knock out the PRB1 gene in the genome of strain D1 to obtain strain T2; S3. Knock out the PRC1 gene in the genome of strain D1 to obtain strain T3; S4. Knock out the YPS1 gene in the genome of strain D1 to obtain strain T4; S5. Knock out the YAP3 gene in the genome of strain D1 to obtain strain T5; S6. Knock out the CPS1 gene in the genome of strain D1 to obtain strain T6; S7. Knock out the PEA1 gene in the genome of strain D1 to obtain strain T7; 11) Perform one or more of the following operations on the yeast strain T1 obtained in step 10): S1. Knock out the OPI1 gene in the genome of strain T1 to obtain strain T8; S2. Knock out the RTN1 gene in the genome of strain T1 to obtain strain T9; S3. Knock out the YOP1 gene in the genome of strain T1 to obtain strain T10; S4. Transform the RTN2 expression vector into T1 to obtain strain G1; 12) The RTN2 expression vector was transformed into strain T8 to obtain strain T12.

7. The construction method according to claim 6, characterized in that: The human serum albumin expression vector mentioned in step 1) is an expression vector containing nucleic acid with the sequence shown in SEQ ID NO.2; Step 11) The RTN2 expression vector mentioned in S4 is an expression vector containing a nucleic acid with the sequence shown in SEQ ID NO. 15; The RTN2 expression vector mentioned in step 12) is an expression vector containing nucleic acid with the sequence shown in SEQ ID NO.

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8. The use of the engineered Saccharomyces cerevisiae according to any one of claims 1 to 5 in the production of recombinant human serum albumin and / or in increasing the secretory expression level of recombinant human serum albumin.

9. The application according to claim 8, characterized in that... The process includes the following steps: fermenting the engineered Saccharomyces cerevisiae of claim 8 in a fermentation medium, centrifuging to remove the cells after fermentation, and obtaining a supernatant containing recombinant human serum albumin.

10. The application according to claim 9, characterized in that: The fermentation medium is SD-2×SCAA+Ura medium or SD-2×SCAA-Ura medium; The fermentation conditions are 20–40 °C and 100–300 rpm for 84–108 h.

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