Kluyveromyces marxianus engineering strain for improving the expression level of recombinant glycoprotein secretion and application thereof

Abstract

The present application belongs to the field of microbial genetic engineering technology, and specifically relates to a Kluyveromyces marxianus engineering strain for improving the secretion and expression level of recombinant glycoprotein and application thereof. The engineering strain is obtained by using gene editing technology to introduce a human UGGT1, UGGT2, SEP15 and EDEM2 expression frame into Kluyveromyces marxianus based on FIM1DU as a starting strain, and introducing a point mutation into an endogenous alpha-glucosidase II beta subunit Gtb1. UGGT1 and UGGT2 are used to introduce a reglycosylation module, and UGGT1 and / or UGGT2 are further enhanced when used in combination with SEP15; the introduction of EDEM2 helps to reduce the degradation rate of intracellular glycoprotein; and the point mutation of Gtb1 can slow down the deglycosylation process. The strain constructed by the above single modification or combined modification can improve the expression and secretion level of the recombinant glycoprotein without affecting the biomass accumulation of the strain, indicating that the engineering strain has important application value for the production of recombinant glycoprotein.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering technology, specifically relating to a Kluyveromyces martensii engineered strain for improving the expression and secretion levels of recombinant glycoproteins and its application. Background Technology

[0002] Efficient expression and secretion of recombinant glycoproteins are a crucial technological foundation in biopharmaceutical and industrial enzyme engineering (O'Flaherty et al., Expert Rev Proteomics. 2018, 15(1):13-29). For antibody fragments, industrial enzymes, and other recombinant secretory proteins with N-glycosylation modifications, their expression levels depend not only on transcription and translation efficiency but also on the synergistic quality control between the endoplasmic reticulum's secretion of correctly folded proteins and degradation of misfolded proteins. During folding, the glycan state of a glycoprotein largely determines whether it enters the secretion pathway or the degradation pathway. Therefore, the glycoprotein quality control system is a key factor limiting efficient glycoprotein secretion (Piirainen MA et al., Front Mol Biosci. 2022, 9:910709).

[0003] In mammalian cells, the folding of N-glycosylated proteins depends on the classic Calnexin / Calreticulin (CNX / CRT) cycle. This system achieves repeated error correction of protein folding intermediates by regulating the removal and reglucosylation of N-glycosylated glucose residues. Specifically, a protein carrying the Glc1Man9GlcNAc2 glycan enters the CNX / CRT cycle, is recognized by CNX / CRT, and folds with the help of cofactors. During folding, α-glucosidase II removes the terminal glucose residue of Glc1Man9GlcNAc2, converting it to Man9GlcNAc2. If the glycoprotein is in a correctly folded state, it is recognized by the COPII complex and transported to the Golgi apparatus. If the glycoprotein is misfolded, it is recognized by UDP-glucose:glycoprotein glucosyltransferase (UGGT). UGGT reglucosylates Man9GlcNAc2 back to Glc1Man9GlcNAc2. After being recognized by CNX / CRT, the substrate re-enters the cycle. Glycoproteins that fail to reach their normal conformation after multiple cycles undergo mannose residue pruning by rate-limiting enzymes EDEM2, EDEM1, and EDEM3, generating glycan signals recognized by the ER-associated degradation (ERAD) pathway, and are then degraded. This mechanism enables mammalian cells to process structurally complex glycoprotein substrates more efficiently (Ninagawa et al., eLife. 2024,12:RP93117).

[0004] In contrast, the quality control mechanism of yeast glycoproteins is relatively simple. Most industrial yeasts lack a functional UGGT-mediated reglucosylation mechanism, so their N-glycosylated proteins, after being modified by Man9GlcNAc2, cannot be reglycosylated and re-enter the folding cycle. In addition, yeast lacks a rate-limiting enzyme with the same function as EDEM2 during mannose trimming, so misfolded proteins are more easily guided to degradation pathways. Compared with the human glycoprotein quality control system of repeated folding and careful degradation, the yeast quality control system is more like a one-time quality control process. Due to the above differences, human glycoproteins expressed in yeast systems are prone to premature degradation or misaggregation due to insufficient folding time or folding ability, thus affecting secretion yield (Ninagawa et al., Biochim Biophys Acta Gen Subj. 2021,1865(3):129812).

[0005] Kluyveromyces marxianus (Km) possesses rapid growth, strong thermostability, a wide range of carbon source utilization, and GRAS (Generally Recognized as Safe) properties, making it a yeast expression host with significant industrial potential. Compared to Saccharomyces cerevisiae, Km has a shorter mannose chain, providing a foundation for further glycosylation engineering. However, Km lacks a complete reglucosylation quality control cycle, and the folding repair capacity of human glycoproteins in this host remains limited.

[0006] Currently, the main strategies for improving the secretory expression levels of recombinant glycoproteins include promoter and codon optimization, signal peptide modification, secretion pathway modification, and knockout of host proteases. However, a strategy for introducing a reglucosylation module into the endoplasmic reticulum to regulate the deglucosylation rate is lacking. Therefore, it is necessary to construct a human-mimicking glycoprotein quality control system in the Km, with a reglucosylation module at its core, to improve the secretion levels of human glycoproteins. Summary of the Invention

[0007] The purpose of this invention is to provide a Kluyveromyces martensii engineered strain that can effectively improve the secretory expression level of recombinant glycoproteins and its application, so as to overcome the problem that some human glycoproteins are prone to low secretion yield when recombinantly expressed in yeast due to insufficient folding time and capacity and premature shunting degradation.

[0008] The Kluyveromyces martensii engineered strain provided by this invention, which improves the secretory expression level of recombinant glycoproteins, is constructed by using Kluyveromyces martensii FIM1ΔU as the starting strain and employing CRISPR-Cas9 gene editing technology to introduce one or more of the following proteins into Kluyveromyces martensii (Km): human reglucosylation-related proteins UGGT1 and / or UGGT2, co-cofactor SEP15, misfolded glycoprotein shunting-related regulatory protein EDEM2, and combining them with the regulation of endogenous Gtb1 activity.

[0009] The starting strain FIM1ΔU was constructed by knocking out the URA3 gene in Kluyveromyces marxianus FIM-1 (deposited at the China General Microbiological Culture Collection Center, accession number CGMCC No. 10621, deposited on March 13, 2015).

[0010] The Kluyveromyces martensii engineered strain accordingly includes one or more of the following protein expression cassettes: UGGT1 expression cassette, UGGT2 expression cassette, SEP15 expression cassette, EDEM2 expression cassette and / or GTB1-E110A mutant.

[0011] In this invention, genes UGGT1, UGGT2, SEP15, and EDEM2 have the nucleotide sequences shown in Gene ID 56886, 129-4653bp, Gene ID 55757, 84-4548 bp, Gene ID 9403, 96-495 bp, and Gene ID 55741, 66-1734bp, respectively.

[0012] In this invention, corresponding expression cassettes were constructed to locate and express human reglucosylation-related proteins UGGT1 and UGGT2, co-helper SEP15, and misfolded glycoprotein shunting-related regulatory factor EDEM2 in the Km endoplasmic reticulum.

[0013] The UGGT1 expression cassette includes the KmPDI1 signal peptide sequence, the UGGT1 coding sequence (129-4653 bp) with the signal peptide removed, the HDEL sequence, and the Saccharomyces cerevisiae ADH1 promoter and terminator, denoted as P. ScADH1 - SP KmPDI1 -UGGT1-HDEL-T ScADH1 .

[0014] The UGGT2 expression cassette includes the KmPDI1 signal peptide sequence, the UGGT2 coding sequence (84-4548 bp) with the signal peptide removed, and the Saccharomyces cerevisiae GPD1 promoter and terminator, denoted as P. ScGPD1 -SP KmPDI1 -UGGT2-T ScGPD1 .

[0015] The SEP15 expression cassette includes the KmPDI1 signal peptide sequence, the SEP15 coding sequence (96–495 bp) with the signal peptide removed and the 96-position U mutated to C, the HDEL sequence, and the Saccharomyces cerevisiae CYS3 promoter and terminator. It is denoted as P. ScCYS3 -SP KmPDI1 -SEP15-U96C-HDEL-T ScCYS3 .

[0016] The EDEM2 expression cassette includes the KmPDI1 signal peptide sequence, the EDEM2 sequence (66-1734 bp) with the signal peptide removed, HDEL, the Saccharomyces cerevisiae TEF1 promoter, and the terminator. It is denoted as P. ScTEF1 -SP KmPDI1 -EDEM2-HDEL-T ScTEF1 .

[0017] Furthermore, the constructed expression cassettes UGGT1, UGGT2, SEP15, and EDEM2 were integrated into the sg1, sg4, sg7, and sg19 sites of the FIM1ΔU genome using CRISPR-Cas9-mediated gene editing (Zhou et al., Commun Biol. 2024, 2;7(1):797), respectively, to construct strains denoted as Km-UGGT1, Km-UGGT2, Km-SEP15, and Km-EDEM2. The strain constructed by integrating all four expression cassettes into the genome was denoted as Km-UGGT1-UGGT2-SEP15-EDEM2.

[0018] Furthermore, it also includes strains constructed by integrating the UGGT2 expression cassette into the sg4 site of the Km-UGGT1 genome, or integrating the UGGT1 expression cassette into the sg1 site of the Km-UGGT2 genome, and denoted as Km-UGGT1-UGGT2.

[0019] The Km-UGGT1, Km-UGGT2, Km-UGGT1-UGGT2 and Km-EDEM2 strains provided by this invention secrete IgG Fc fragments (IgG Fc) expressing human IgG antibodies at yields that are 55.3%, 32%, 107% and 52.3% higher than those of the FIM1ΔU (WT) strain, respectively.

[0020] Furthermore, it also includes using Km-SEP15 as the starting strain, introducing UGGT1, UGGT2, or UGGT1+UGGT2 expression cassettes respectively to construct strains, denoted as Km-UGGT1-SEP15, Km-UGGT2-SEP15, and Km-UGGT1-UGGT2-SEP15.

[0021] The experimental results showed that the IgG Fc secretion expression levels of strains Km-UGGT1-SEP15, Km-UGGT2-SEP15 and Km-UGGT1-UGGT2-SEP15 were increased by approximately 99.2%, 38.6% and 153.3% respectively compared with the control strain.

[0022] This invention also provides an engineered method for improving the expression level of recombinant glycoproteins by regulating the activity of the endogenous α-glucosidase II β subunit Gtb1 in Km to modulate the rate of endoplasmic reticulum deglucosylation.

[0023] Specifically, an amino acid substitution is performed at the catalytic active site of the endogenous α-glucosidase II β subunit Gtb1 to reduce the deglucosylation rate and prolong the protein folding time of translation; preferably, the amino acid substitution is to replace the glutamic acid (E) at position 110 of Gtb1 with alanine (A) to obtain a mutant, denoted as GTB1-E110A.

[0024] Furthermore, in this invention, using a CRISPR-Cas9-mediated gene editing method, the α-glucosidase II β subunit encoding gene GTB1 in the FIM1ΔU strain is first knocked out, and then the GTB1-E110A gene is inserted in situ to construct the engineered strain, denoted as Km-GTB1-E110A. Similarly, the GTB1 gene in the Km-UGGT1-UGGT2-SEP15-EDEM2 strain is first knocked out, and then the GTB1-E110A gene is inserted in situ to construct the engineered strain, denoted as Km-AY.

[0025] Experimental results showed that the Km-GTB1-E110A and Km-AY strains produced 30.4% and 202.5% higher yields of human IgG Fc than the WT strain, respectively. Among them, the highest yield of IgG Fc in Km-AY during fed-batch fermentation reached 1.36 g / L, while that in WT was only about 0.45 g / L.

[0026] The present invention also relates to a CRISPR-Cas9 gene editing system for the engineered strain Km-AY, comprising an ARS1-Cas9 / gRNA vector.

[0027] The ARS1-Cas9 / gRNA vector contains a Cas9 expression cassette and a gRNA expression cassette; the gRNA nucleotide sequences for UGGT1, UGGT2, SEP15, EDEM2 and GTB1 are shown in SEQ ID NO: 1-12.

[0028] The present invention also provides the application of the engineered strain of *Kluyveromyces martensii* in the soluble secretory expression of recombinant glycoproteins.

[0029] The recombinant glycoproteins include, but are not limited to, antibody IgG Fc fragments and other secretory proteins containing N-glycosylation sites.

[0030] Specifically, it includes the following steps:

[0031] (1) Codon optimization of the IgG Fc coding sequence;

[0032] (2) The IgG Fc coding sequence is cloned into a yeast secretory expression vector, the vector containing a yeast autonomous replication sequence, a promoter, a signal peptide, a terminator and a selection marker;

[0033] (3) The expression vector was introduced into the Kluyveromyces martensii strain;

[0034] (4) The target glycoprotein was obtained by culturing and collecting the supernatant under fed-batch fermentation conditions.

[0035] The engineered strains of this invention were obtained by introducing human UGGT1, UGGT2, SEP15, or EDEM2 expression cassettes into *Kluyveromyces martensii* and introducing a point mutation in the endogenous α-glucosidase II β subunit Gtb1. UGGT1 and UGGT2 are used to introduce the reglucosylation module; SEP15 plays an enhancing role when combined with UGGT1, UGGT2, or UGGT1+UGGT2; the introduction of EDEM2 helps to reduce the degradation rate of intracellular glycoproteins; and the point mutation in Gtb1 slows down the deglucosylation process. The engineered strains constructed by the above single or combined modifications can improve the expression and secretion levels of recombinant glycoproteins to varying degrees. Under fed-batch fermentation conditions, the IgG Fc protein yield of the Km-AY strain reached 1.36 g / L, approximately 3 times higher than that of the control strain FIM1ΔU. The above modifications did not significantly affect the biomass accumulation of the strains, indicating that these engineered strains have application value in the production of recombinant glycoproteins.

[0036] The Km engineered strain provided by this invention can be used for the expression of different types of recombinant glycoproteins such as antibody drugs and Fc fusion proteins, and has good prospects for industrial application. Attached Figure Description

[0037] Figure 1 The effect of UGGT1 on IgG Fc expression levels was investigated. A. After 24 hours of fermentation in a fermenter, SDS-PAGE analysis and grayscale quantification of IgG Fc expression levels in Km-UGGT1 and WT (FIM1ΔU) were performed. Arrows indicate the Fc bands used for quantification.

[0038] Figure 2 The effect of UGGT2 on IgG Fc expression levels was investigated. A. After 24 hours of fermentation in a fermenter, SDS-PAGE analysis and grayscale quantification of IgG Fc expression levels in Km-UGGT2 and WT (FIM1ΔU) were performed. Arrows indicate the Fc bands used for quantification.

[0039] Figure 3The effect of SEP15 and its combination with UGGT on IgG Fc expression levels was investigated. After 24 hours of fermentation, SDS-PAGE analysis and grayscale quantification of IgG Fc expression levels in Km-SEP15 (A), Km-UGGT1-SEP15 (B), Km-UGGT2-SEP15 (C), Km-UGGT1-UGGT2, and Km-UGGT1-UGGT2-SEP15 (D) compared to WT (FIM1ΔU) were performed. Arrows indicate the Fc bands used for quantification.

[0040] Figure 4 The effect of EDEM2 on IgG Fc expression levels was investigated. A. After 24 hours of fermentation in a fermenter, SDS-PAGE analysis and grayscale quantification of IgG Fc expression levels in Km-EDEM2 and WT (FIM1ΔU) were performed. Arrows indicate the Fc bands used for quantification.

[0041] Figure 5 The effect of GTB1-E110A on IgG Fc expression levels was investigated. A. After 24 hours of fermentation in a fermenter, SDS-PAGE analysis and grayscale quantification of IgG Fc expression levels in Km-GTB1-E110A and WT (FIM1ΔU) were performed. Arrows indicate the Fc bands used for quantification.

[0042] Figure 6 The data represents the IgG Fc expression level of the final engineered strain Km-AY. Specifically, A. SDS-PAGE analysis and grayscale quantification of IgG Fc expression levels in the final engineered strain Km-AY and WT(FIM1ΔU) after 24 hours of fermenter culture. BC. SDS-PAGE analysis and grayscale quantification of deglycosylated IgG Fc expression levels in Km-AY and WT(FIM1ΔU) at different time points during fermenter culture. β-lactoglobulin was used as the standard protein for quantification. The arrows indicate the Fc bands used for quantification.

[0043] Figure 7 This is a schematic diagram of the reconstructed glycosylation process of the present invention. Detailed Implementation

[0044] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0045] Example 1: Construction of an engineered strain including the human UGGT1 expression cassette and its effect on the expression level of secreted glycoproteins in Kluyveromyces martensii.

[0046] In this embodiment, CRISPR-Cas9 genome editing technology was used to construct an engineered strain expressing human UGGT1 in Kluyveromyces martensii (Km) and its effect on the expression level of secreted glycoprotein was examined.

[0047] The starting strain was FIM1ΔU. The UGGT1 expression cassette was: 178bp upstream homologous arm of sg1 - P. ScADH1 -SP KmPDI1 -UGGT1-HDEL-T ScADH1 -sg1 downstream homologous arm 420bp. To construct the above expression cassette, using HeLa cell cDNA as a template, the UGGT1 coding sequence, after removing the natural signal peptide and the natural C-terminal endoplasmic reticulum resident signal coding sequence, was amplified. The primers used for PCR amplification were UGGT1-F1 (SEQ ID No. 14) and UGGT1-R1 (SEQ ID No. 15). After PCR, the amplification products were analyzed by 1% agarose gel electrophoresis, and the target fragment was recovered. The PCR product was recovered using the SanPrep column DNA gel recovery kit (Sangon Biotech, catalog number B518131-0050), and the specific operation was performed according to the instructions. The composition of the UGGT1 fragment obtained by amplification using the above primers is: P ScADH1 3' end 25bp homologous arm-SP KmPDI1 -UGGT1-HDEL-T ScADH1 A 25bp homologous arm at the 5' end. The donor backbone plasmid for genome integration is pDonor-1. pDonor-1 is constructed by inserting a 178bp homologous arm upstream of the sg1 site into the cloning site of pMD18-T (Takara, catalog number 6011). ScADH1 T ScADH1The pDonor-1 backbone fragment and the 420bp downstream homologous arm of the sg1 site were also identified. Using pDonor-1 as a template, a linearized backbone fragment was obtained by circular PCR amplification using primers UGGT1-F2 (SEQ ID No. 16) and UGGT1-R2 (SEQ ID No. 17). After PCR, the amplification products were analyzed by 1% agarose gel electrophoresis, and the target fragment was recovered. PCR product recovery was also performed using the SanPrep DNA gel extraction kit (Sangon Biotech, catalog number B518131-0050). The linearized pDonor-1 backbone fragment and the aforementioned UGGT1 fragment were homologously ligated using the Gibson Assembly ligation system (ABclonal, catalog number RK21020) to obtain a donor plasmid containing the UGGT1 expression cassette. Transformation into *E. coli* and sequencing verification yielded the donor plasmid pDonor-5. The obtained pDonor-5 was used as the donor vector for subsequent site-directed integration of the human UGGT1 expression cassette into the genome. To achieve stable expression of the UGGT1 expression cassette in the Km genome, site-specific integration was performed using the CRISPR-Cas9 system, with the genomically safe site sg1 selected as the integration site. A gRNA sequence targeting the sg1 site was designed, in which gRNA-101F (SEQ ID No. 1) and gRNA-101R (SEQ ID No. 2) were annealed to form a double-stranded DNA fragment. This double-stranded fragment was ligated into an ARS1-Cas9 / gRNA vector (LHZ531) (Shi et al., Biotechnol Biofuels. 14;14(1):236) linearized by LguI restriction, transformed into E. coli, and sequenced to obtain the recombinant CRISPR-101 plasmid. Using pDonor-5 as a template, the donor fragment for genome integration was amplified using primers UGGT1-F3 (SEQ ID No. 18) and UGGT1-R3 (SEQ ID No. 19), named 101-Donor. The 101-Donor includes: the 178 bp upstream homologous arm of sg1. ScADH1 -SP KmPDI1 -UGGT1-HDEL-T ScADH1The downstream homologous arm of sg1 is 420 bp. The recombinant CRISPR-101 plasmid and 101-Donor were co-transformed into FIM1ΔU, and candidate integration clones were screened on SC-Ura plates. After the transformants grew, colony PCR was performed to identify the integration site using the site-outer primers UGGT1-F4 (SEQ ID No. 20) and UGGT1-R4 (SEQ ID No. 21). Clones with the expected band size were further sequenced to confirm that the UGGT1 expression cassette had been correctly integrated into the sg1 site. Clones with correct sequenced integration were streaked on YPD plates containing 5-fluoroorotic acid (5-FOA, 1.5 g / L) to lose the CRISPR-101 plasmid. Single clones were then sequenced to confirm that the UGGT1 expression cassette remained stably integrated into the genome after the loss of the CRISPR-101 plasmid. The correctly integrated engineered strain was named Km-UGGT1.

[0048] To investigate the effect of UGGT1 expression on the expression level of secreted glycoproteins, the coding sequence of the human IgG antibody Fc fragment obtained from gene synthesis (SEQ ID No. 13) was constructed into the yeast secretory expression vector pUKD-132-C29T (pZP33) (Zhou Jetal. Biotechnol Biofuels.) to obtain the secretory expression plasmid pExp-IgG Fc. Specifically, the IgG Fc coding sequence was ligated into the pUKD-132-C29T vector, which had been linearized by double digestion with SmaI and NotI, using the Gibson Assembly scarless ligation system to construct pExp-IgG Fc. Subsequently, pExp-IgG Fc was transformed into the Km-UGGT1 strain and the control FIM1ΔU, respectively, to obtain the expression strains Km-UGGT1-IgG Fc and FIM1ΔU-IgG Fc. After activation on SC-Ura solid plates, the strains were inoculated into 50 mL of SM seed medium and cultured at 30 ℃ and 220 rpm for 12 h. The SM seed culture medium consisted of: (NH4)2SO4 5 g / L, glucose 10 g / L, MgSO4·7H2O 0.5 g / L, KH2PO4 3 g / L, trace elements 2 mL / L, and vitamins 1 mL / L. The seed culture was then inoculated into a 1.5 L fermenter for fermentation, with a working volume of 600 mL. The SM fermentation medium consisted of: (NH4)2SO4 12 g / L, glucose 10 g / L, MgSO4·7H2O 7 g / L, KH2PO4 20 g / L, trace elements 16.7 mL / L, and vitamins 2 mL / L. During fermentation, glucose was added continuously, the temperature was controlled at 30 ℃, the pH was automatically controlled at 5.5 using ammonia, the sterile filtered air aeration rate was 0.6 L / min, and the stirring speed was controlled by dissolved oxygen cascade within the range of 300-1000 rpm. After 24 h of fermentation, the culture supernatant was collected by centrifugation, and the expression of secreted proteins was analyzed by SDS-PAGE. ImageJ software was used for grayscale scanning and quantitative analysis of the target bands. The SDS-PAGE results showed ( Figure 1 Compared with the control strain FIM1ΔU-IgG Fc, the IgG Fc band in the supernatant of Km-UGGT1-IgG Fc was significantly enhanced. Gray-scale scanning quantification results showed that the secretory expression level of IgG Fc in Km-UGGT1-IgG Fc was approximately 55.3% higher than that in the control. These results indicate that site-specific integration and expression of human UGGT1 in Km can increase the expression level of secretory glycoproteins, suggesting that the introduction of the reglucosylation module helps to promote enhanced secretion levels.

[0049] Example 2: Construction of an engineered strain including the human UGGT2 expression cassette and its effect on the expression level of secreted glycoproteins in Kluyveromyces martensii.

[0050] In this embodiment, human UGGT2 expression engineered strains were constructed in Kluyveromyces martensii (Km) using CRISPR-Cas9 genome editing technology, and their effect on the expression level of secreted glycoproteins was examined.

[0051] The starting strain was the same as in Example 1, FIM1ΔU. The UGGT2 expression cassette was: 357 bp upstream homologous arm of sg4 - P ScGPD1 -SP KmPDI1 -UGGT2-T ScGPD1 -sg4 downstream homologous arm 367 bp. To construct the above expression cassette, the UGGT2 coding sequence with the natural signal peptide removed was amplified using HeLa cell cDNA as a template. The primers used for PCR amplification were UGGT2-F1 (SEQ ID No. 22) and UGGT2-R1 (SEQ ID No. 23). After PCR, the amplification products were analyzed by 1% agarose gel electrophoresis, and the target fragment was recovered. The PCR product was recovered using the SanPrep column DNA gel recovery kit (Sangon Biotech, catalog number B518131-0050), and the specific operation was performed according to the instructions. The composition of the UGGT2 fragment obtained by amplification using the above primers is: P ScGPD1 3' end 25bp homologous arm-SP KmPDI1 -UGGT2-T ScGPD1A 25bp homologous arm at the 5' end. The donor backbone plasmid for genome integration was pDonor-2. pDonor-2 was constructed by inserting a 357bp homologous arm upstream of the sg4 site, the *Saccharomyces cerevisiae* GPD1 promoter, the *Saccharomyces cerevisiae* GPD1 terminator, and a 367bp homologous arm downstream of the sg4 site into the pMD18-T cloning site. Using pDonor-2 as a template, a linearized backbone fragment was obtained by circular PCR amplification using primers UGGT2-F2 (SEQ ID No. 24) and UGGT2-R2 (SEQ ID No. 25). After PCR, the amplification products were analyzed by 1% agarose gel electrophoresis, and the target fragment was recovered. PCR product recovery was also performed using the SanPrep DNA Gel Extraction Kit (Sangon Biotech, catalog number B518131-0050). The linearized pDonor-2 backbone fragment and the aforementioned UGGT2 amplified fragment were ligated using the Gibson Assembly ligation system (ABclonal, catalog number RK21020) for homologous recombination to obtain a donor plasmid containing the UGGT2 expression cassette. After transformation into *E. coli* and sequencing verification, the donor plasmid pDonor-6 was obtained. pDonor-6 was used as the human UGGT2 expression donor vector for subsequent site-directed integration into the genome. To achieve stable expression of the UGGT2 expression cassette in the Km genome, site-directed integration was performed using the CRISPR-Cas9 system, selecting the genomically safe site sg4 as the integration site. A gRNA sequence targeting the sg4 site was designed, where gRNA-201F (SEQ ID No. 3) and gRNA-201R (SEQ ID No. 4) were annealed to form a double-stranded DNA fragment. This double-stranded fragment was ligated into an ARS1-Cas9 / gRNA vector linearized by LguI digestion, transformed into *E. coli*, and sequenced to obtain the recombinant CRISPR-201 plasmid. Using pDonor-6 as a template, a donor fragment for genome integration was obtained by amplification using primers UGGT2-F3 (SEQ ID No. 26) and UGGT2-R3 (SEQ ID No. 27), named 201-Donor. 201-Donor contains: a 357 bp upstream homologous arm of sg4. ScGPD1 -SP KmPDI1 -UGGT2-T ScGPD1The downstream homologous arm of sg4 is 367 bp. The recombinant CRISPR-201 plasmid and 201-Donor were co-transformed into FIM1ΔU, and candidate integration clones were screened on SC-Ura plates. After the transformants grew, colony PCR was performed to identify the integration site using the outer primers UGGT2-F4 (SEQ ID No. 28) and UGGT2-R4 (SEQ ID No. 29). Clones with the expected band size were further sequenced to confirm that the UGGT2 expression cassette had been correctly integrated into the sg4 site. Clones with correct sequenced integration were streaked on YPD plates containing 5-fluoroorotic acid (5-FOA, 1.5 g / L) to lose the CRISPR-201 plasmid. Single clones were then sequenced to confirm that the UGGT2 expression cassette remained stably integrated into the genome after the loss of the CRISPR-201 plasmid. The correctly integrated engineered strain was named Km-UGGT2.

[0052] To investigate the effect of UGGT2 expression on the expression level of secreted glycoproteins, the IgG Fc secretion expression plasmid pExp-IgG Fc obtained in Example 1 was transformed into the Km-UGGT2 strain and the control strain FIM1ΔU, respectively, to obtain the expression strains Km-UGGT2-IgG Fc and FIM1ΔU-IgG Fc. After activation on SC-Ura solid plates, these strains were cultured under the same fermentation conditions as described in Example 1. After 24 h of fermentation, the culture supernatant was collected by centrifugation, and the expression of secreted proteins was analyzed by SDS-PAGE. ImageJ software was used for grayscale scanning and quantitative analysis of the target bands. The SDS-PAGE results showed ( Figure 2 Compared with the control strain FIM1ΔU-IgG Fc, the IgG Fc band in the supernatant of Km-UGGT2-IgG Fc was significantly enhanced. Gray-scale scanning quantification results showed that the secretory expression level of IgG Fc in Km-UGGT2-IgG Fc was approximately 32% higher than that in the control. These results indicate that site-specific integration and expression of human UGGT2 in Km can also increase the expression level of secretory glycoproteins, suggesting that UGGT2 also has an independent secretion-promoting effect.

[0053] Example 3: Construction of an engineered strain including the human SEP15 expression cassette and its effect on the expression level of secreted glycoproteins in *Kluyveromyces martensii* when combined with UGGT.

[0054] In this embodiment, human SEP15 expression engineered strains were constructed in Kluyveromyces martensii (Km) using CRISPR-Cas9 genome editing technology, and their effect on the expression level of secreted glycoproteins was examined.

[0055] The starting strain was the same as in Example 1, FIM1ΔU. The SEP15 expression cassette was: 120 bp upstream homologous arm of sg7 - P. ScCYS3 -SP KmPDI1 -SEP15-U96C-HDEL-T ScCYS3 -sg7 downstream homologous arm 407 bp. To construct the above expression cassette, using HeLa cell cDNA as a template, the SEP15 coding sequence, after removing the natural signal peptide and the natural C-terminal endoplasmic reticulum resident signal coding sequence, was amplified. The primers used for PCR amplification were SEP15-F1 (SEQ ID No. 30) and SEP15-R1 (SEQ ID No. 31). After PCR, the amplification products were analyzed by 1% agarose gel electrophoresis, and the target fragment was recovered. The PCR product was recovered using the SanPrep column DNA gel recovery kit (Sangon Biotech, catalog number B518131-0050), and the specific operation was performed according to the instructions. The composition of the SEP15 fragment obtained by amplification using the above primers is: P ScCYS3 3' end 25bp homologous arm-SP KmPDI1 -SEP15-HDEL-T ScCYS3A 25 bp homologous arm at the 5' end. The donor backbone plasmid for genome integration was pDonor-3. pDonor-3 was constructed by inserting a 120 bp homologous arm upstream of the sg7 site, the *Saccharomyces cerevisiae* CYS3 promoter, the *Saccharomyces cerevisiae* CYS3 terminator, and a 407 bp homologous arm downstream of the sg7 site into the pMD18-T cloning site. Using pDonor-3 as a template, a linearized backbone fragment was obtained by circular PCR amplification using primers SEP15-F2 (SEQ ID No. 32) and SEP15-R2 (SEQ ID No. 33). After PCR, the amplification products were analyzed by 1% agarose gel electrophoresis, and the target fragment was recovered. PCR product recovery was also performed using the SanPrep DNA Gel Extraction Kit (Sangon Biotech, catalog number B518131-0050). The linearized pDonor-3 backbone fragment and the aforementioned SEP15 amplified fragment were ligated using the Gibson Assembly ligation system (ABclonal, catalog number RK21020) for homologous recombination to obtain a donor plasmid containing the SEP15 expression cassette. Transformation into *E. coli* and sequencing verification yielded the donor plasmid pDonor-7. Further, to obtain a SEP15 variant that can be stably translated in yeast and to avoid the decoding restriction of selenocysteine ​​(U), pDonor-7 was subjected to site-directed mutagenesis, replacing selenocysteine ​​(U) at amino acid position 96 of SEP15 with cysteine ​​(C), resulting in the SEP15-U96C donor plasmid pDonor-9. The site-directed mutagenesis was achieved using circular PCR with primers SEP15-F3 (SEQ ID No. 34) and SEP15-R3 (SEQ ID No. 35). The circular PCR product was digested with DpnI to remove the template and then transformed into *E. coli*. Sequencing confirmed that the U96C mutation was correct and that there were no other unexpected mutations. To achieve stable expression of the SEP15 expression cassette in the Km genome, site-specific integration was performed using the CRISPR-Cas9 system, with the genomically safe site sg7 selected as the integration site. A gRNA sequence targeting the sg7 site was designed, in which gRNA-302F (SEQ ID No. 5) and gRNA-302R (SEQ ID NO: 6) were annealed to form a double-stranded DNA fragment. This double-stranded fragment was ligated into an ARS1-Cas9 / gRNA vector linearized by LguI digestion, transformed into *E. coli*, and sequenced to obtain the recombinant CRISPR-302 plasmid. Using pDonor-9 as a template, the donor fragment for genome integration was amplified using primers SEP15-F4 (SEQ ID No. 36) and SEP15-R4 (SEQ ID No. 37), named 302-Donor. The 302-Donor includes: the 120 bp-P upstream homologous arm of sg7. ScCYS3 -SPKmPDI1 -SEP15-U96C-HDEL-T ScCYS3 The downstream homologous arm of sg7 is 407 bp. The recombinant CRISPR-302 plasmid and 302-Donor were co-transformed into FIM1ΔU, and candidate integration clones were screened on SC-Ura plates. After the transformants grew, colony PCR was performed to identify the integration site using the outer primers SEP15-F5 (SEQ ID No. 38) and SEP15-R5 (SEQ ID No. 39). Clones with the expected band size were further sequenced to confirm that the SEP15-U96C expression cassette had been correctly integrated into the sg7 site. Clones with correct sequenced integration were streaked on YPD plates containing 5-fluoroorotic acid (5-FOA, 1.5 g / L) to lose the CRISPR-302 plasmid. Single clones were then sequenced to confirm that the SEP15-U96C expression cassette remained stably integrated into the genome after the loss of the CRISPR-302 plasmid. The engineered strain with correct integration was named Km-SEP15.

[0056] Based on this, further targeted integration was performed according to the methods of Examples 1 and 2 to construct a combined engineered strain:

[0057] (1) Using Km-SEP15 as the starting strain, the UGGT1 expression cassette was site-directedly integrated into the sg1 site according to the method in Example 1 to obtain the engineered strain Km-UGGT1-SEP15;

[0058] (2) Using Km-SEP15 as the starting strain, the UGGT2 expression cassette was site-directedly integrated into the sg4 site according to the method in Example 2 to obtain the engineered strain Km-UGGT2-SEP15;

[0059] (3) Further construct a three-gene combination strain: Based on Km-UGGT1-SEP15, integrate the UGGT2 expression cassette into the sg4 site according to the method of Example 2; or based on Km-UGGT2-SEP15, integrate the UGGT1 expression cassette into the sg1 site according to the method of Example 1 to obtain the engineered strain Km-UGGT1-UGGT2-SEP15.

[0060] (4) Using Km-UGGT1 as the starting strain, the UGGT2 expression cassette was site-directedly integrated into the sg4 site according to the method in Example 2 to obtain the engineered strain Km-UGGT1-UGGT2;

[0061] After the above integrations were screened and confirmed by SC-Ura plates and sequencing, they were then streaked on YPD plates containing 5-fluoroorotic acid (5-FOA, 1.5 g / L) to lose the corresponding CRISPR plasmids, thus obtaining stable engineered strains. The outer primers used for the relevant integration identification sites were as described in Example 1 or Example 2, respectively.

[0062] To detect the effect of the above-mentioned strains on the expression level of secreted glycoproteins, the IgG Fc secretory expression plasmid pExp-IgG Fc obtained in Example 1 was transformed into Km-SEP15, Km-UGGT1-SEP15, Km-UGGT2-SEP15, Km-UGGT1-UGGT2, Km-UGGT1-UGGT2-SEP15, and the control strain FIM1ΔU, respectively, to obtain the corresponding IgG Fc expression strains. The fermentation and detection methods were the same as in Example 1. After 24 h of fermentation, the culture supernatant was collected by centrifugation, and the expression of secreted proteins was analyzed by SDS-PAGE. ImageJ software was used for grayscale scanning and quantitative analysis of the target bands. The results showed ( Figure 3 Compared with the control strain FIM1ΔU-IgG Fc, the IgG Fc secretion expression level of Km-SEP15-IgG Fc showed no significant change; however, in the UGGT-containing combination strains, IgG Fc secretion expression was significantly enhanced. Specifically, the IgG Fc secretion expression level of Km-UGGT1-SEP15-IgG Fc was approximately 99.2% higher than the control. The IgG Fc secretion expression level of Km-UGGT2-SEP15-IgG Fc was approximately 38.6% higher than the control, the IgG Fc secretion expression level of Km-UGGT1-UGGT2-IgG Fc was approximately 107.0% higher than the control, and the IgG Fc secretion expression level of Km-UGGT1-UGGT2-SEP15-IgG Fc was approximately 153.3% higher than the control. These results indicate that SEP15 expression alone has a limited effect on enhancing secretion, but it can synergistically enhance the expression level of secreted glycoproteins with UGGT1 and / or UGGT2.

[0063] Example 4: Construction of an engineered strain including the human EDEM2 expression cassette and its effect on the expression level of secreted glycoproteins in Kluyveromyces martensii.

[0064] In this embodiment, human EDEM2 expression engineered strains were constructed in Kluyveromyces martensii (Km) using CRISPR-Cas9 genome editing technology, and their effect on the expression level of secreted glycoproteins was examined.

[0065] The starting strain was the same as in Example 1, FIM1ΔU. The EDEM2 expression cassette was: sg19 upstream homologous arm 124 bp-P ScTEF1 -SPKmPDI1 -EDEM2-HDEL-T ScTEF1 -sg19 downstream homologous arm 351 bp. To construct the above expression cassette, using HeLa cell cDNA as a template, the EDEM2 coding sequence, after removing the natural signal peptide and the natural C-terminal endoplasmic reticulum resident signal coding sequence, was amplified. The primers used for PCR amplification were EDEM2-F1 (SEQ ID No. 40) and EDEM2-R1 (SEQ ID No. 41). After PCR, the amplification products were analyzed by 1% agarose gel electrophoresis, and the target fragment was recovered. The PCR product was recovered using the SanPrep column DNA gel recovery kit (Sangon Biotech, catalog number B518131-0050), and the specific operation was performed according to the instructions. The composition of the EDEM2 fragment obtained by amplification using the above primers is: P ScTEF1 3' end 25bp homologous arm-SP KmPDI1 -EDEM2-HDEL-T ScTEF1 A 25bp homologous arm at the 5' end. The donor backbone plasmid for genome integration was pDonor-4. pDonor-4 was constructed by inserting a 124bp homologous arm upstream of the sg19 site, the *Saccharomyces cerevisiae* TEF1 promoter, the *Saccharomyces cerevisiae* TEF1 terminator, and a 351bp homologous arm downstream of the sg19 site into the pMD18-T cloning site. Using pDonor-4 as a template, a linearized backbone fragment was obtained by circular PCR amplification using primers EDEM2-F2 (SEQ ID No. 42) and EDEM2-R2 (SEQ ID No. 43). After PCR, the amplification products were analyzed by 1% agarose gel electrophoresis, and the target fragment was recovered. PCR product recovery was also performed using the SanPrep DNA Gel Extraction Kit (Sangon Biotech, catalog number B518131-0050). The linearized pDonor-4 backbone fragment and the aforementioned EDEM2 amplified fragment were ligated using the Gibson Assembly ligation system (ABclonal, catalog number RK21020) for homologous recombination to obtain a donor plasmid containing the EDEM2 expression cassette. After transformation into *E. coli*, sequencing confirmed the presence of the donor plasmid pDonor-8. The resulting pDonor-8 was used as the human EDEM2 expression donor vector for subsequent site-directed genome integration.

[0066] To achieve stable expression of the EDEM2 expression cassette in the Km genome, site-specific integration was performed using the CRISPR-Cas9 system, with the genomically safe site sg19 selected as the integration site. A gRNA sequence targeting the sg19 site was designed, in which gRNA-702F (SEQ ID No. 7) and gRNA-702R (SEQ ID No. 8) were annealed to form a double-stranded DNA fragment. This double-stranded fragment was ligated into an ARS1-Cas9 / gRNA vector linearized by LguI digestion, transformed into *E. coli*, and sequenced to obtain the recombinant CRISPR-702 plasmid. Using pDonor-8 as a template, primers EDEM2-F3 (SEQ ID No. 44) and EDEM2-R3 (SEQ ID No. 45) were used to amplify the donor fragment for genome integration, named 702-Donor. 702-Donor contains: a 124 bp upstream homologous arm of sg19. ScTEF1 -SP KmPDI1 -EDEM2-HDEL-T ScTEF1 The downstream homologous arm of sg19 is 351 bp. The recombinant CRISPR-702 plasmid and 702-Donor were co-transformed into FIM1ΔU, and candidate integration clones were screened on SC-Ura plates. After the transformants grew, colony PCR was performed to identify the integration site using the outer primers EDEM2-F4 (SEQ ID No. 46) and EDEM2-R4 (SEQ ID No. 47). Clones with the expected band size were further sequenced to confirm that the EDEM2 expression cassette had been correctly integrated into the sg19 site. Clones with correct sequenced integration were streaked on YPD plates containing 5-fluoroorotic acid (5-FOA, 1.5 g / L) to lose the CRISPR-702 plasmid. Single clones were then sequenced to confirm that the EDEM2 expression cassette remained stably integrated into the genome after the loss of the CRISPR-702 plasmid. The correctly integrated engineered strain was named Km-EDEM2.

[0067] To investigate the effect of EDEM2 expression on the expression levels of secreted glycoproteins, the IgG Fc secretory expression plasmid pExp-IgG Fc obtained in Example 1 was transformed into the Km-EDEM2 strain and the control strain FIM1ΔU, respectively, to obtain the expression strains Km-EDEM2-IgG Fc and FIM1ΔU-IgG Fc. After activation on SC-Ura solid plates, these strains were cultured under the same fermentation conditions as described in Example 1. After 24 h of fermentation, the culture supernatant was collected by centrifugation, and the expression of secreted proteins was analyzed by SDS-PAGE. ImageJ software was used for grayscale scanning and quantitative analysis of the target bands. The results showed ( Figure 4Compared with the control strain FIM1ΔU-IgG Fc, the IgG Fc band in the supernatant of Km-EDEM2-IgG Fc was significantly enhanced. Gray-scale scanning quantification results showed that the secretory expression level of IgG Fc in Km-EDEM2-IgG Fc was approximately 52.3% higher than that in the control strain. These results indicate that site-specific integration and expression of human EDEM2 in Km can increase the expression level of secreted glycoproteins, suggesting that the introduction of a human rate-limiting enzyme that regulates the degradation of misfolded glycoproteins can reduce misdegradation of glycoproteins and promote their secretory expression.

[0068] Example 5: Construction of an engineered strain including the GTB1-E110A mutant and its effect on the expression level of secreted glycoproteins in Kluyveromyces martensii.

[0069] In this embodiment, CRISPR-Cas9 genome editing technology was used to construct the Gtb1 catalytic site mutant E110A in Kluyveromyces martensii (Km) and its effect on the expression level of secreted glycoprotein was examined.

[0070] The starting strain was the same as in Example 1, FIM1ΔU. First, a Gtb1 deletion donor plasmid was constructed. Using FIM1ΔU genomic DNA as a template, the upstream 500 bp and downstream 1000 bp homologous arm sequences of the GTB1 gene were amplified by PCR. The primers used were GTB1-F1 (SEQ ID No. 48) / GTB1-R1 (SEQ ID No. 49) (upstream homologous arm) and GTB1-F2 (SEQ ID No. 50) / GTB1-R2 (SEQ ID No. 51) (downstream homologous arm), respectively. After PCR, the amplification products were analyzed by 1% agarose gel electrophoresis, and the target fragment was recovered. PCR product recovery was performed using a SanPrep column-based DNA gel recovery kit (Sangon Biotech, catalog number B518131-0050), following the manufacturer's instructions. The recovered upstream and downstream homologous arm fragments of GTB1 were ligated into the pMD18-T vector using the Gibson Assembly ligation system (ABclonal, catalog number RK21020) to obtain the GTB1 knockout donor plasmid pDonor-20. The recombinant plasmid was identified by E. coli colony PCR and sequenced to confirm its correct sequence. The primers were GTB1-F1 (SEQ ID No. 48) and GTB1-R2 (SEQ ID No. 51). Using pDonor-20 as a template, the donor fragment for knocking out the GTB1 ORF was amplified using GTB1-F3 (SEQ ID No. 52) and GTB1-R3 (SEQ ID No. 53), named GTB1-KO-Donor. GTB1-KO-Donor comprises: a 500 bp upstream homologous arm of GTB1 and a 1000 bp downstream homologous arm of GTB1.

[0071] Further, to construct the Gtb1 catalytic site mutant, using FIM1ΔU genomic DNA as a template, a DNA fragment containing the GTB1 coding sequence and its upstream 500 bp and downstream 1000 bp homologous arms was amplified using GTB1-F1 and GTB1-R2. After PCR product detection and recovery by 1% agarose gel electrophoresis, homologous recombination was performed with the pMD18-T vector using the Gibson Assembly traceless ligation system to obtain an intermediate vector containing the complete Gtb1 expression unit. Subsequently, site-directed mutagenesis was performed using circular PCR to replace glutamic acid (E) with alanine (A) at amino acid position 110 of Gtb1, using primers GTB1-F4 (SEQ ID No. 54) and GTB1-R4 (SEQ ID No. 55). The circular PCR product was digested with DpnI to remove the template and transformed into *E. coli*. Sequencing confirmed the presence of the E110A mutant donor plasmid, named pDonor-21. Using pDonor-21 as a template, donor fragments for integrating the GTB1-E110A mutant were amplified using GTB1-F3 and GTB1-R3, and named GTB1-KI-Donor. GTB1-KI-Donor comprises: a 500 bp upstream homologous arm of GTB1, a 1000 bp downstream homologous arm of GTB1, and GTB1-E110A.

[0072] To construct the Km-GTB1-E110A in situ mutant strain, a two-step CRISPR editing strategy was employed. First, a Gtb1 deletion strain was constructed. gRNA sequences targeting the GTB1 ORF were designed; the sequences of gRNA-GTB1-KO-F and gRNA-GTB1-KO-R are shown in SEQ ID No. 9 and SEQ ID No. 10, respectively. The annealed double-stranded DNA fragment was ligated into the ARS1-Cas9 / gRNA vector, which was linearized by LguI digestion. After transformation into *E. coli*, sequencing verification yielded the recombinant CRISPR-GTB1-KO plasmid. This plasmid, along with GTB1-KO-Donor, was co-transformed into FIM1ΔU, and candidate transformants were screened on SC-Ura plates. After the transformants grew, colony PCR was performed using primers outside the site (primers: GTB1-F3 / GTB1-R3), and sequencing verification confirmed the acquisition of the Gtb1 deletion strain, named Km-gtb1Δ. Clones that were correctly sequenced were streaked onto YPD plates containing 5-fluoroorotic acid (5-FOA, 1.5 g / L) to remove the CRISPR-GTB1-KO plasmid. In the second step, the GTB1-E110A mutant was integrated orally into Km-gtb1Δ. gRNA sequences targeting GTB1 in situ integration were designed; the sequences of gRNA-GTB1-KI-F and gRNA-GTB1-KI-R are shown in SEQ ID No. 11 and SEQ ID No. 12, respectively. The annealed double-stranded DNA fragment was ligated into the linearized ARS1-Cas9 / gRNA vector digested with LguI, transformed into *E. coli*, and sequenced to obtain the recombinant CRISPR-GTB1-KI plasmid. This plasmid and GTB1-KI-Donor were co-transformed into Km-gtb1Δ, and candidate transformants were screened on SC-Ura plates. After the transformants grew, colony PCR was performed using primers outside the original GTB1 site (GTB1-F3 and GTB1-R3), and sequencing confirmed that GTB1-E110A had been correctly integrated into the original GTB1 site. The correctly sequenced clones were streaked onto YPD plates containing 5-fluoroorotic acid (5-FOA, 1.5 g / L) to remove the CRISPR-GTB1-KI plasmid. Subsequently, single-clone engineered strains were sequenced to confirm that the genome still stably carried the GTB1-E110A mutation after the loss of the CRISPR-GTB1-KI plasmid, resulting in the engineered strain Km-GTB1-E110A. This two-step editing strategy achieved precise replacement of the GTB1 site.

[0073] To investigate the effect of the GTB1-E110A mutation on the expression level of secreted glycoproteins, the IgGFc secretory expression plasmid pExp-IgG Fc obtained in Example 1 was transformed into Km-GTB1-E110A and the control strain FIM1ΔU, respectively, to obtain expression strains Km-GTB1-E110A-IgG Fc and FIM1ΔU-IgG Fc. The fermentation and detection methods were the same as in Example 1. After 24 h of fermentation, the culture supernatant was collected by centrifugation, and the expression of secreted proteins was analyzed by SDS-PAGE. ImageJ software was used for grayscale scanning and quantitative analysis of the target bands. The results showed ( Figure 5 Compared with the control strain FIM1ΔU-IgG Fc, the IgG Fc band in the supernatant of Km-GTB1-E110A-IgG Fc was significantly enhanced. Quantitative analysis using grayscale scanning showed that its IgG Fc secretion expression level was approximately 30.4% higher than that of the control strain. These results suggest that moderately reducing the α-glucosidase II deglucosylation process using the GTB1-E110A mutant may help prolong the substrate's folding window in the endoplasmic reticulum, thereby improving the maturation efficiency and export level of secreted glycoproteins.

[0074] Example 6: Construction of an engineered strain including a combination of human cofactors and its effect on the expression level of secreted glycoproteins in Kluyveromyces martensii.

[0075] Based on the Km-UGGT1-UGGT2-SEP15 strain constructed in Example 3, the EDEM2 expression cassette was further integrated into the genomic safe site sg19 according to the method described in Example 4, resulting in the four-factor combined engineered strain Km-UGGT1-UGGT2-SEP15-EDEM2. Subsequently, following the two-step CRISPR editing strategy described in Example 5, the endogenous GTB1 gene ORF was first knocked out, and then the GTB1-E110A donor sequence was introduced via homologous recombination to achieve in situ substitution, ultimately obtaining the five-factor combined final engineered strain Km-AY. The above gene integration and site substitution were verified by colony PCR outside the site and confirmed by Sanger sequencing. Clones with correct sequencing verification were cultured on YPD plates containing 5-fluoroorotic acid (5-FOA, 1.5 g / L) to lose the corresponding CRISPR plasmids, resulting in genetically stable combined engineered strains.

[0076] To evaluate the effect of multi-module collaborative reconstruction on the expression level of secreted glycoproteins, the IgGFc secretory expression plasmid pExp-IgG Fc obtained in Example 1 was transformed into the Km-AY strain and the control strain FIM1ΔU, respectively, to obtain expression strains Km-AY-IgG Fc and FIM1ΔU-IgG Fc. The fermentation conditions and secretory protein detection methods were the same as in Example 1. After 24 h of fermentation, the culture supernatant was collected by centrifugation, and the expression of secreted proteins was analyzed by SDS-PAGE, using β-lactoglobulin as a quantitative standard. ImageJ software was used for grayscale scanning quantitative analysis of the target bands; simultaneously, the change in IgG Fc yield during fermentation was continuously monitored. The results showed ( Figure 6 Compared with the control strain FIM1ΔU-IgG Fc, Km-AY-IgG Fc showed an increase of approximately 202.5% in IgG Fc secretion expression at 24 h of fermentation, and reached its highest yield of approximately 1.36 g / L at 60 h.

[0077] The results from Examples 1–5 show that Km-AY exhibits a more significant enhancement effect compared to single-factor engineered strains and some combined engineered strains. These results indicate that by combining the introduction of reglucosylation elements and their cofactors, misfolded glycoprotein shunting-related regulatory factors, and deglucosylation rate regulators, the dynamic balance between protein folding, quality control, and shunting degradation in the endoplasmic reticulum can be synergistically optimized, thereby significantly improving the expression level of secretory glycoproteins by Km. This multi-factor combined modification strategy provides an effective engineering solution for enhancing the secretory expression of recombinant glycoproteins in yeast cell factories.

Claims

1. A Kluyveromyces macrocephala engineered strain that enhances the secretory expression level of recombinant glycoproteins, characterized in that, Using Kluyveromyces martensii FIM1ΔU as the starting strain, one or more of the following proteins were introduced into Kluyveromyces martensii (Km) using CRISPR-Cas9 gene editing technology: human reglucosylation-related proteins UGGT1 and / or UGGT2, co-helper SEP15, and misfolded glycoprotein shunting-related regulatory protein EDEM2, and combined with the regulation of endogenous Gtb1 activity to construct the strain. The starting strain FIM1ΔU was constructed by knocking out the URA3 gene in Kluyveromyces martensii FIM-1, which is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 10621.

2. The engineered strain of *Kluyveromyces martensii* according to claim 1, characterized in that, Accordingly, it includes one or more of the following protein expression cassettes: UGGT1 expression cassette, UGGT2 expression cassette, SEP15 expression cassette, EDEM2 expression cassette and / or GTB1-E110A mutant.

3. The engineered strain of *Kluyveromyces martensii* according to claim 2, characterized in that, The protein expression cassette was constructed to locate and express human reglucosylation-related proteins UGGT1 and UGGT2, co-helper SEP15, and misfolded glycoprotein shunting-related regulator EDEM2 in the Km endoplasmic reticulum, wherein: The UGGT1 expression cassette includes the KmPDI1 signal peptide sequence, the UGGT1 coding sequence (129-4653bp) with the signal peptide removed, the HDEL sequence, and the Saccharomyces cerevisiae ADH1 promoter and terminator, denoted as P. ScADH1 -SP KmPDI1 -UGGT1-HDEL-T ScADH1 ; The UGGT2 expression cassette includes the KmPDI1 signal peptide sequence, the UGGT2 coding sequence (84-4548 bp) with the signal peptide removed, and the Saccharomyces cerevisiae GPD1 promoter and terminator, denoted as P. ScGPD1 -SP KmPDI1 -UGGT2-T ScGPD1 ; The SEP15 expression cassette includes the KmPDI1 signal peptide sequence, the SEP15 coding sequence (96–495 bp) with the signal peptide removed and the 96-position U mutated to C, the HDEL sequence, the Saccharomyces cerevisiae CYS3 promoter and terminator, denoted as P. ScCYS3 -SP KmPDI1 -SEP15-U96C-HDEL-T ScCYS3 ; The EDEM2 expression cassette includes the KmPDI1 signal peptide sequence, the EDEM2 sequence (66-1734 bp) with the signal peptide removed, HDEL, the Saccharomyces cerevisiae TEF1 promoter, and the terminator, denoted as P. ScTEF1 -SP KmPDI1 -EDEM2-HDEL-T ScTEF1 .

4. The engineered strain of *Kluyveromyces martensii* according to claim 3, characterized in that, It also includes strains constructed by integrating the UGGT1, UGGT2, SEP15 and EDEM2 expression cassettes into the sg1, sg4, sg7 and sg19 sites of the FIM1ΔU genome using CRISPR-Cas9-mediated gene editing technology, denoted as Km-UGGT1, Km-UGGT2, Km-SEP15 and Km-EDEM2, respectively, and strains constructed by integrating all four expression cassettes into the genome simultaneously, denoted as Km-UGGT1-UGGT2-SEP15-EDEM2.

5. The engineered strain of *Kluyveromyces martensii* according to claim 4, characterized in that, It also includes strains constructed by integrating the UGGT2 expression cassette into the sg4 site of the Km-UGGT1 genome, or integrating the UGGT1 expression cassette into the sg1 site of the Km-UGGT2 genome, denoted as Km-UGGT1-UGGT2.

6. The engineered strain of *Kluyveromyces martensii* according to claim 4, characterized in that, It also includes strains constructed by introducing UGGT1, UGGT2, or both UGGT1 and UGGT2 expression cassettes, starting with Km-SEP15. These strains are referred to as Km-UGGT1-SEP15, Km-UGGT2-SEP15, and Km-UGGT1-UGGT2-SEP15, respectively.

7. The engineered strain of *Kluyveromyces martensii* according to claim 4, characterized in that, This also includes CRISPR-Cas9-mediated gene editing technology, first knocking out the GTB1 gene encoding the α-glucosidase II β subunit of FIM1ΔU, and then inserting the GTB1-E110A gene in situ to construct an engineered strain, denoted as Km-GTB1-E110A; similarly, first knocking out GTB1 in the Km-UGGT1-UGGT2-SEP15-EDEM2 strain, and then inserting the GTB1-E110A gene in situ to construct an engineered strain, denoted as Km-AY; wherein, GTB1-E110A is a mutant obtained by replacing the catalytic active site of the Km endogenous α-glucosidase II β subunit Gtb1 with amino acid, wherein the amino acid replacement is replacing glutamic acid (E) at position 110 of Gtb1 with alanine (A).

8. The use of the engineered strain of *Kluyveromyces martensii* as described in any one of claims 1-7 in the soluble secretory expression of recombinant glycoproteins.

9. The application according to claim 8, characterized in that, The recombinant glycoprotein includes antibody IgG Fc fragments and other secretory proteins containing N-glycosylation sites.

10. The application according to claim 9, characterized in that, Includes the following steps: (1) Codon optimization of the IgG Fc coding sequence; (2) The IgG Fc coding sequence is cloned into a yeast secretory expression vector, the vector containing a yeast autonomous replication sequence, a promoter, a signal peptide, a terminator and a selection marker; (3) The expression vector was introduced into the Kluyveromyces martensii strain; (4) The target glycoprotein was obtained by culturing and collecting the supernatant under fed-batch fermentation conditions.

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