A pichia pastoris engineering strain overexpressing alg14 with improved expression and secretion capacity and a method for improving the expression amount of exogenous proteins

Abstract

The present application relates to the field of agricultural biotechnology, and in particular to a Pichia pastoris engineering strain with improved expression and secretion capacity and overexpression of ALG14 and a method for improving the expression amount of exogenous proteins, and the main technical problem to be solved by the present application is to overcome the deficiencies of the prior art, and to provide a strategy for improving the expression of exogenous proteins by Pichia pastoris, and to improve the enzyme production capacity of Pichia pastoris by overexpression of the AGL14 gene of Pichia pastoris itself.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a Pichia pastoris engineered strain that overexpresses ALG14 with enhanced expression and secretion capabilities, and a method for increasing the expression level of exogenous proteins. Background Technology

[0002] Pichia pastoris ( Komagataella phaffii As a methyltrophic yeast, Pichia pastoris is a highly efficient heterologous protein expression system. However, the expression levels of heterologous enzymes or functional proteins in Pichia pastoris are still generally low and cannot meet the needs of industrial production.

[0003] SEM1 is the 26S proteasome complex subunit SEM1. This gene in Pichia pastoris is only mentioned in genome sequence annotation.

[0004] ALG7 is the Pichia pastoris UDP-N-acetylglucosamine-1-phosphotransferase. UDP-N-acetylglucosamine-phosphopolyterpenoid N-acetylglucosamine phosphotransferase is an enzyme that acts on the biosynthetic pathway of polyterpenoid-linked oligosaccharides. These oligosaccharides are glycan precursors for protein asparagine (N-) glycosylation. Assembly of polyterpenoid-linked oligosaccharides begins on the cytoplasmic side of the endoplasmic reticulum membrane and is completed within its lumen.

[0005] ALG14 is a component of Pichia pastoris UDP-GlcNAc transferase, involved in protein N-glycosylation; it is essential for the second step of the polyterpene-linked oligosaccharide pathway; and it anchors the catalytic subunit ALG13 to the endoplasmic reticulum.

[0006] To address key challenges in heterologous protein expression, methods such as screening target genes, optimizing codons, selecting expression vectors, and replacing promoters and signal peptides are commonly used to increase protein expression levels. However, these methods are highly specific to the expressed protein sequence. Therefore, obtaining universally applicable components or methods for enhancing protein expression is of great significance. Summary of the Invention

[0007] The purpose of this invention is to provide a method for improving the protein expression and secretion capacity of Pichia pastoris cells.

[0008] Another object of the present invention is to provide a method for increasing the expression level of exogenous proteins in Pichia pastoris.

[0009] Another object of the present invention is to provide a modified Pichia pastoris engineered strain.

[0010] The method for improving the protein expression and secretion capacity of Pichia pastoris cells according to the present invention includes the step of overexpressing ALG14 factor in Pichia pastoris cells, wherein the amino acid sequence of ALG14 factor is shown in SEQ ID NO:7.

[0011] The method for improving protein expression and secretion capacity of Pichia pastoris cells according to the present invention further includes the step of overexpressing the encoding gene of SEM1 factor in Pichia pastoris cells, wherein the amino acid sequence of SEM1 factor is shown in SEQ ID NO:5.

[0012] According to the method for improving the protein expression and secretion capacity of Pichia pastoris cells of the present invention, the nucleotide sequence of the gene encoding the SEM1 factor is shown in SEQ ID NO:1, and the nucleotide sequence of the gene encoding the ALG14 factor is shown in SEQ ID NO:3.

[0013] The method for improving protein expression and secretion capacity of Pichia pastoris cells according to the present invention includes the steps of constructing recombinant expression vectors containing the coding genes of SEM1 factor and ALG14 factor, respectively.

[0014] According to the method for improving the protein expression and secretion capacity of Pichia pastoris cells of the present invention, wherein the nucleotide sequence of the highly efficient constitutive promoter is shown in SEQ ID NO:4.

[0015] The Pichia pastoris engineered strain with enhanced protein expression and secretion capacity according to the present invention comprises an overexpressed ALG14 factor, wherein the amino acid sequence of the ALG14 factor is shown in SEQ ID NO:7.

[0016] The Pichia pastoris engineered strain with enhanced protein expression and secretion capacity according to the present invention further includes an overexpressed gene encoding the SEM1 factor, the amino acid sequence of which is shown in SEQ ID NO:5.

[0017] The method for increasing the expression level of exogenous proteins in Pichia pastoris according to the present invention includes the following steps:

[0018] Overexpression of ALG14 factor in Pichia pastoris, wherein the amino acid sequence of ALG7 factor is shown in SEQ ID NO:7; and

[0019] Expressing the gene encoding a foreign protein in Pichia pastoris.

[0020] The method for increasing the expression level of exogenous proteins in Pichia pastoris according to the present invention further includes the step of overexpressing the encoding gene of SEM1 factor in Pichia pastoris, wherein the amino acid sequence of SEM1 factor is shown in SEQ ID NO:5.

[0021] According to the method for increasing the expression level of exogenous proteins in Pichia pastoris according to the present invention, the nucleotide sequence of the gene encoding the SEM1 factor is shown in SEQ ID NO:1, and the nucleotide sequence of the gene encoding the ALG14 factor is shown in SEQ ID NO:3.

[0022] The method for increasing the expression level of exogenous proteins in Pichia pastoris according to the present invention includes the steps of constructing recombinant expression vectors containing the coding genes of SEM1 factor and ALG14 factor, respectively.

[0023] According to the method of the present invention for increasing the expression level of exogenous proteins in Pichia pastoris, during the construction of the recombinant expression vector, the coding genes of the SEM1 factor and the ALG14 factor are regulated by a highly efficient constitutive promoter.

[0024] According to the method for increasing the expression level of exogenous proteins in Pichia pastoris according to the present invention, the nucleotide sequence of the highly efficient constitutive promoter is shown in SEQ ID NO:4.

[0025] The method for increasing the expression level of exogenous proteins in Pichia pastoris according to the present invention includes, but is not limited to, xylanase, cellulase, mannanase, glucose oxidase, protease, phytase, and saccharifying enzyme.

[0026] The main technical problem solved by this invention is to overcome the shortcomings of the prior art and provide a strategy to improve the expression of exogenous proteins in Pichia pastoris by overexpressing Pichia pastoris' own ALG14 to enhance the enzyme production capacity of Pichia pastoris.

[0027] Overexpression of ALG14 in this invention increases xylanase production by 13.5 times, cellulase production by 1.5 times, mannanase production by 3.64 times, glucose oxidase production by 1.7 times, phytase production by 2.63 times, glucoamylase production by 3.12 times, and protease production by 1.33 times; co-expression of SEM1 / ALG14 increases xylanase production by 15.6 times.

[0028] Overexpression of ALG14 in this invention can increase xylanase production by 13.5 times, cellulase production by 1.5 times, mannanase production by 3.64 times, glucose oxidase production by 1.7 times, phytase production by 2.63 times, glucoamylase production by 3.12 times, and protease production by 1.33 times. Attached Figure Description

[0029] Figure 1The comparison of the proteome and transcriptome of SEM1, ALG7, and ALG14 factors in xylanase-high and low-expression strains is shown.

[0030] Figure 2 The effects of overexpression of SEM1, ALG7, and ALG14 on the growth rate of Pichia pastoris strains were shown.

[0031] Figure 3 The changes in enzyme activity of Pichia pastoris strains after introducing plasmids overexpressing SEM1, ALG7, and ALG14 were shown.

[0032] Figure 4 This shows the transcriptional levels of essential genes for cell wall synthesis in Pichia pastoris cells after overexpression of SEM1, ALG7, and ALG14, respectively.

[0033] Figure 5 The expression changes of core genes maintaining endoplasmic reticulum homeostasis were shown in strains that overexpressed ALG7 and strains that overexpressed ALG14.

[0034] Figure 6 This shows the changes in xylanase activity after co-expression of SEM1 / ALG7 and SEM1 / ALG14 in Pichia pastoris strains. Detailed Implementation

[0035] The experimental materials and reagents involved in the following examples include:

[0036] Strains and vectors: Pichia pastoris expression vector pPIC9 and strains GS115 ;

[0037] Enzymes and other biochemical reagents: endonucleases, ligases;

[0038] Culture medium:

[0039] BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (v / v).

[0040] BMMY medium: except that 0.5% methanol is used instead of glycerol, all other components are the same as BMGY medium.

[0041] YPD medium: 1% yeast extract, 2% peptone, 2% glucose.

[0042] The following examples involve Pichia pastoris (Pichia pastoris) Komagataella phaffii The secretion and expression-related factors SEM1, ALG7, and ALG14 were isolated from [the organism]. The encoding genes for SEM1, ALG7, and ALG14 were cloned using PCR. sem1, alg7, alg14The full lengths of their sequences are 369bp, 1401bp, and 645bp, respectively.

[0043] in, sem1 The nucleotide sequence is shown in SEQ ID NO: 1.

[0044] SEQ ID NO:1:

[0045] ATGCGCGAAAACCTGGACACTATCAGAAACTCAAAGAAATACTTGAAGCTCTCTCCCTTGATTTATTCTGTCTTATTAACAACCAAATTCAAAATGTCTACAGAGAACAAAGCAGAGACAAAACAAACCAACTTCAAAGCTTTGGAAGAGGATGACGAGTTTGAAGATTTCCCCGTTCAAGGTAA GTTATCAGCAGTGTCAGATCGAGAAGTGGATCAACTAACGACTGAAATAGATTGGCCAAATGACAAGACCATCACAGCTGAAAAACAAGAGCATCTTTGGGATGAGAGCTGGAATGATGACGATAACGAAGATGATTTCACCAAAAGATTGAGACAAGAACTCGCAAACGCTGAAGCAAAGAAA.

[0046] alg7 The nucleotide sequence is shown in SEQ ID NO: 2.

[0047] SEQ ID NO: 2:

[0048]

[0049] alg14 The nucleotide sequence is shown in SEQ ID NO:3.

[0050] SEQ ID NO. 3:

[0051] .

[0052] This invention utilizes a highly efficient expression promoter to achieve overexpression of the target gene. The highly efficient expression promoter is shown in SEQ ID NO:4. This promoter is the pGAP promoter, which is a commonly used highly efficient constitutive promoter in Pichia pastoris. Overexpression of SEM1, ALG7, and ALG14 genes does not affect the growth rate of the strain, and effectively enhances the expression levels of various industrial enzymes such as xylanase, cellulase, mannanase, and glucose oxidase.

[0053] pGAP promoter, SEQ ID NO: 4:

[0054] TTTTTGTAGAAATGTCTTGGTGTCCTCGTTCCAATCAGGTAGCCATCTCTGAAATATCTGGCTCCGTTGCAACTCCGAACGACCTGCTGGCACGTAACATTTTCTCCGGGGTAAAACTTAAATGTGGAGTAATGGGACCAGAAACGTCTCTTCCCTTCTCTCTCCTTCCACCGCCCGTTACCGTCCCTAGGAAATTTTACTCTGCTGGAGAGCTTCTTCTACGGCCCCTTGCAGCAATGCTCTTCCCAGCATTACGTTGCGGGTAAAACGGAGGTCGTGTACCCGACCTAGCAGCCCAGGGATGGAAAAGTCCCGGCCGTCGCTGGCATTAATAGCGGGCGGACGCATGTCTTGAGATTGTTGGAAACCACCAGAATCGAATATAAAAGGCGAACACCTTTCCCAATTTTGGTTTCTCCTGACCCAAAGACTTTAAATTTTATTTATTTGTCCCTATTTCAATCAATTGAACAACTAT。

[0055] The amino acid sequence of SEM1 is shown in SEQ ID NO:5,

[0056] SEQ ID NO:5:

[0057] MRENLDTIRNSKKYLKLSPLIYSVLLTTKFKMSTENKAETKQTNFKALEEDDEFEDFPVQGKLSAVSDREVDQLTTEIDWPNDKTITAEKQEHLWDESWNDDDNEDDFTKRLRQELANAEAKK;

[0058] The amino acid sequence of ALG7 is shown in SEQ ID NO. 6,

[0059] SEQ ID NO:6:

[0060] MQQLPKIGLLAVSMALICHTYSPLQPIQSSIGFAVLGYLLSDYLIPATAPYFIKIGLFGKDLSKKDKPVIPETIGIIPAVVYLFIMFSFIPFMFFKFLVVDTSGGGSRDTGVDLETA DSNYFPHNKLSSYLSGILSLESMVLLGLLDDLFDIRWRHKFFLPAIAAIPLLIVYYVDFGVTHILIPTFIKNIFGFEAVSIDLGALYYGYMAAVAIFCPNSINILAGINGLEVGQSV VLAVLLLLNDFCYLIPASLRSTPAYETHLMSTCILVPFLGVSLSLLKFNWYPAKVFVGDTFCYFSGMVFAFVGISGHFSKTLLLFFLPQIFNFVYSIPQLFGLVECPRHRLPRFNEE DNMMYPSHAVFKKRLPKLIEKGMLILEALGLLEVVKEEMKTDNKTEIIIKECSNFTLINLVLVWFGPMREDRLCFVILLLQFSIGLISLVARHTIAALLFGYDNLSIFNLSILSSN.

[0061] The amino acid sequence of ALG14 is shown in SEQ ID NO: 7.

[0062] SEQ ID NO:7:

[0063] MTTSIYCLLALLISLTLVVIRVIFCVPFCRLQDVSPVLDKPLSVLILLGSGGHTGEMLNILSQLDHKFKYSFIVQSNDESSVLRLEKSQVKGTVYTVPRARNVGDGLL RSIQGTLKCWLGTMKVLVFDKKWKEGNIPSVLLVNGPGSCVPLAYSIVVLNILGLASARIIYMESLTRVNELSLSGKLLYLVADRFVVQWPELAQKYRRTEYHGILV.

[0064] Note: Molecular biology experimental methods not specifically described in the following examples were performed in accordance with the specific methods listed in J. Sambrook's "Molecular Cloning: A Laboratory Manual" (3rd Edition), or according to the kit and product instructions.

[0065] This application is based on two existing Pichia pastoris strains expressing xylanase in the laboratory, with an expression level difference of more than 10-fold between the two strains. These strains were cultured, and samples were taken at 0, 24, and 72 hours after methanol induction for transcriptome and proteome sequencing. The results showed that the three genes mentioned above were positively correlated with high xylanase expression in both the proteome and transcriptome; that is, the transcriptional and protein levels of these three genes were higher in the xylanase-high expression strains than in the xylanase-low expression strains.

[0066] Example 1: Cloning of SEM1, ALG7, and ALG14 factor encoding genes derived from Pichia pastoris (Komagataella phaffii).

[0067] Two Pichia pastoris strains with xylanase expression levels differing by more than 10-fold were sampled at 0, 24, and 72 hours after methanol induction for transcriptome and proteome sequencing. SEM1, ALG7, and ALG14 factors were positively correlated with high xylanase expression in both the proteome and transcriptome; that is, the transcriptional and protein levels of these three genes were higher in the xylanase-high expression strains than in the xylanase-low expression strains. Figure 1 ).

[0068] Extraction of Pichia pastoris Komagataella phaffii Genomic DNA. According to... SEM1 Conserved sequences were used to design and synthesize the corresponding primers F1 and R1 (the underlined parts represent the vector backbone).

[0069] F1: 5'- GAACAACTATCGCCACC ATGCGCGAAAACCTGGACACTATCAG -3' (SEQ ID NO:8);

[0070] R1: 5'- TCATCATCATCCTTGTAATC TTTCTTTGCTTCAGCGTTTGCGAG-3' (SEQ ID NO:9).

[0071] by Komagataella phaffii Total DNA was used as a template for PCR amplification. A fragment of approximately 400 bp was obtained, which was recovered, ligated into a vector, and then sequenced.

[0072] according to ALG7 Conserved sequences were used to design and synthesize the corresponding primers F2 and R2:

[0073] F2:5'- GAACAACTATCGCCACC ATGCAACAATTACCCAAAATAGGACTATTGGCAG -3' (SEQ IDNO:10);

[0074] R2:5'- TCATCATCATCCTTGTAATCTTGTTCGTTGGAACTCAGTATGCTGAGATTGAAAATAC-3' (SEQ ID NO: 11).

[0075] by Komagataella phaffii Total DNA was used as a template for PCR amplification. A fragment of approximately 1500 bp was obtained, which was recovered, ligated into a vector, and then sequenced.

[0076] according to ALG14 Conserved sequences were used to design and synthesize the corresponding primers F3 and R3:

[0077] F3:5'- GAACAACTATCGCCACC ATGACCACTTCAATTTACTGTCTTTTGGCTTTATTG -3' (SEQ ID NO: 12);

[0078] R3:5'- TCATCATCATCCTTGTAATC GACAAGGATTCCATGGTACTCGGTAC-3' (SEQ ID NO: 13).

[0079] by Komagataella phaffii Total DNA was used as a template for PCR amplification, yielding a fragment of approximately 700 bp. This fragment was then recovered, ligated into a vector, and sent for sequencing.

[0080] Pichia pastoris requires a wet weight of 200 g / L before induction production. Competent Pichia pastoris (CK) cells were prepared, and plasmids overexpressing SEM1, ALG7, and ALG14 were introduced. Three strains successfully overexpressing SEM1, ALG7, and ALG14 were screened and inoculated into YPD medium along with Pichia pastoris (CK). After two 1% inoculum transfers to obtain secondary seed culture, the culture was transferred to a 15L fermenter for fermentation. Wet weight was measured every 6 hours. It was found that, compared to Pichia pastoris (CK), overexpression of SEM1, ALG7, and ALG14 did not affect the growth rate of Pichia pastoris. Figure 2 ).

[0081] Example 2: Expression of exogenous proteins in recombinant strains expressing SEM1, ALG7, and ALG14 factors

[0082] 2.1 Construction of engineered strains

[0083] The overexpression vector PACZ containing the promoter shown in SEQ ID NO: 4 was digested with Sac I, and the gene fragments encoding SEM1 / ALG7 / ALG14 obtained above were ligated to the vector PACZ to obtain recombinant vectors PACZ-SEM1, PACZ-ALG7 and PACZ-ALG14.

[0084] Genes encoding xylanase, cellulase, mannanase, and glucose oxidase were ligated into the EcoRI and NotI sites of the pPIC9 plasmid to obtain exotropic expression plasmids of *Pichia pastoris*. These plasmids were digested with DraI, and the products were recovered and electroporated into GS115 competent cells, then plated into MD plates. Two days later, single clones on the MD plates were cultured using yeast tubes. After methanol induction, the activities of xylanase, cellulase, mannanase, and glucose oxidase in the supernatant were measured, yielding recombinant *Pichia pastoris* strains that correctly expressed xylanase, cellulase, mannanase, and glucose oxidase, respectively. These *Pichia pastoris* strains expressing xylanase, cellulase, mannanase, and glucose oxidase were collected and cultured, and corresponding competent cells were prepared again.

[0085] The overexpression vectors PACZ-SEM1, PACZ-ALG7, and PACZ-ALG14 were digested with enzymes (Pme I), and after recovery, they were electroporated into the Pichia pastoris strains that expressed xylanase, cellulase, mannanase, and glucose oxidase, respectively, to obtain engineered Pichia pastoris strains that simultaneously expressed the relevant enzymes and co-expressed different factors.

[0086] 2.2 Enzyme Activity Analysis

[0087] Xylanase activity assay: Under pH 5.0 and 55℃ conditions, a 1 mL reaction system consisted of 100 μL of appropriately diluted enzyme solution and 900 μL of 1% beech xylan. The reaction was allowed to proceed for 10 min, then 1.5 mL of DNS was added to terminate the reaction, and the mixture was boiled in water for 5 min. After cooling, the OD value was measured at 540 nm. One enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute under given conditions. The xylanase activity of strains overexpressing the target gene was compared with that of strains originally expressing xylanase to obtain the percentage increase in xylanase activity after overexpression.

[0088] Cellulase activity assay: Under pH 4.0 and 70℃ conditions, a 1 mL reaction system consisted of 100 μL of appropriately diluted enzyme solution and 900 μL of 1% CMCNa. The reaction was allowed to proceed for 10 min, then 1.5 mL of DNS was added to terminate the reaction, and the mixture was boiled in water for 5 min. After cooling, the OD value was measured at 540 nm. One unit of enzyme activity (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute under given conditions. Using the strain originally expressing cellulase as a control, the cellulase activity of the strain overexpressing the target gene was compared to obtain the percentage increase in cellulase activity after overexpression.

[0089] Mannanase activity assay: Under pH 5.0 and 70℃ conditions, a 1 mL reaction system consisted of 100 μL of appropriately diluted enzyme solution and 900 μL of 1% CMCNa. The reaction was allowed to proceed for 10 min, then 1.5 mL of DNS was added to terminate the reaction, and the mixture was boiled in water for 5 min. After cooling, the OD value was measured at 540 nm. One enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute under given conditions. Using the strain originally expressing mannanase as a control, the mannanase activity of the strain overexpressing the target gene was compared to obtain the percentage increase in mannanase activity after overexpression.

[0090] Glucose oxidase activity assay: Under pH 6.0 and 30℃ conditions, a 1 mL reaction system consisted of 50 μL of appropriately diluted enzyme solution and 950 μL of mixed substrate. The reaction was allowed to proceed for 3 min, and then 1 mL of 2M H₂SO₄ was added to terminate the reaction. After cooling, the OD value was measured at 540 nm. One enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute under given conditions. Using the strain originally expressing glucose oxidase as a control, the glucose oxidase activity of the strain overexpressing the target gene was compared to obtain the percentage increase in glucose oxidase activity after overexpression.

[0091] Figure 3 In the diagram, CK corresponds to the original Pichia pastoris strains in which various enzyme expression plasmids were introduced. After introducing SEM1, ALG7, and ALG14 plasmids into Pichia pastoris strains that successfully expressed xylanase, cellulase, mannanase, GOD, phytase, saccharifying enzyme, and protease, respectively, the enzyme activity changes of the corresponding strains were detected. The relative enzyme activity of CK was defined as 100%, and a comparative enzyme activity diagram was obtained.

[0092] like Figure 3 As shown, the enzyme activity enhancement effects of overexpressing SEM1, ALG7, and ALG14 factors are illustrated in Pichia pastoris strains expressing xylanase, cellulase, mannanase, and glucose oxidase, respectively. Overexpression of SEM1 increased xylanase production by 10 times, cellulase production by 2.1 times, mannanase production by 3.08 times, glucose oxidase production by 1.87 times, phytase production by 2.05 times, saccharifying enzyme production by 2.04 times, and protease production by 1.31 times.

[0093] Overexpression of ALG7 increased xylanase production by 9.4 times, cellulase production by 1.8 times, mannanase production by 2.8 times, glucose oxidase production by 2.72 times, phytase production by 2.68 times, glucoamylase production by 2.27 times, and protease production by 1.34 times.

[0094] Overexpression of ALG14 increased xylanase production by 13.5 times, cellulase production by 1.5 times, mannanase production by 3.64 times, glucose oxidase production by 1.7 times, phytase production by 2.63 times, glucoamylase production by 3.12 times, and protease production by 1.33 times.

[0095] Example 3: Construction of Pichia pastoris chassis cells with enhanced protein secretion capacity

[0096] 3.1 Overexpression of the SEM1 gene can reduce the transcription level of genes essential for cell wall synthesis, thereby increasing cell wall permeability and facilitating protein secretion.

[0097] Genes 0226 and 0227 are β-1,3-glucanyltransferase genes, essential for cell wall synthesis. For example... Figure 4 As shown, overexpression of SEM1, ALG7, and ALG14 in Pichia pastoris cells reduced the transcription levels of essential genes for cell wall synthesis (0226, 0227), meaning that overexpression of SEM1, ALG7, and ALG14 inhibited the transcription of Pichia pastoris cell wall synthesis genes 0226 and 0227.

[0098] 3.2 Overexpression of ALG7 and ALG14 improves endoplasmic reticulum homeostasis

[0099] OST1 is an oligosaccharide transferase or OST complex that participates in N-glycosylation, ensuring correct protein folding and secretion, and reducing the accumulation of unfolded proteins. HRD1 is an E3 ubiquitin ligase, encoding the HRD1 E3 ubiquitin ligase, a key enzyme in the endoplasmic reticulum homeostasis pathway (ERAD), which mediates the ubiquitination and degradation of misfolded proteins. OST1, by reducing the accumulation of misfolded proteins, works with HRD1 to mediate the degradation of misfolded proteins, thus alleviating endoplasmic reticulum stress.

[0100] HAC1 (UPR transcription factor), co-expression of HAC1i (splice-activated form) can activate the unfolded protein response (UPR), enhance the endoplasmic reticulum protein processing capacity (such as molecular chaperone and folding enzyme expression), relieve endoplasmic reticulum stress, and maintain homeostasis. It is a gene that enhances the endoplasmic reticulum protein processing capacity.

[0101] GSC1 is 1,3-β-glucan synthase; ROT1 is the α subunit of glucosidase II. GSC1 and ROT1 can help proteins fold correctly and reduce misfolded proteins.

[0102] The genes mentioned above are all core genes for maintaining endoplasmic reticulum homeostasis, such as... Figure 5 As shown, in strains that overexpress ALG7 and strains that overexpress ALG14, upregulation of the gene shown can effectively improve endoplasmic reticulum homeostasis.

[0103] In summary, ALG7 and ALG14 can regulate the bacterial cell state from multiple angles and globally, jointly achieving efficient secretion of exoproteins.

[0104] Example 4: Co-expression of SEM1 / ALG7 and SEM1 / ALG14 with ALG7 / AG14 enhances xylanase activity.

[0105] SEM1 / ALG7 and SEM1 / ALG14 were co-expressed with ALG7 / AG14. These two genes were constructed on the same overexpression plasmid, and both genes were simultaneously overexpressed using two expression cassettes. The overexpression was accomplished using the GAP strong promoter and the GCW14 strong promoter, respectively. The effect of co-expression on enhancing xylanase activity was detected. Figure 6 Co-expression of SEM1 / ALG7 increased xylanase production by 12.9 times, co-expression of SEM1 / ALG14 increased xylanase production by 15.6 times, and co-expression of ALG14 / ALG7 increased xylanase production by 21 times.

[0106] The above embodiments are only used to understand the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. A method for improving the expression and secretion capacity of exogenous proteins in Pichia pastoris cells, characterized in that, The method includes the following steps: Overexpression of ALG14 factor in Pichia pastoris cells, wherein the amino acid sequence of ALG14 factor is shown in SEQ ID NO:7, and the exogenous protein is xylanase, cellulase, mannanase, glucose oxidase, phytase or saccharifying enzyme.

2. The method for improving the expression and secretion capacity of exogenous proteins in Pichia pastoris cells according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the ALG14 factor is shown in SEQ ID NO:

3.

3. The method for improving the expression and secretion capacity of exogenous proteins in Pichia pastoris cells according to claim 1, characterized in that, The method further includes the step of overexpressing the encoding gene of the SEM1 factor in Pichia pastoris cells, the amino acid sequence of which is shown in SEQ ID NO:

5.

4. The method for improving the expression and secretion of exogenous proteins in Pichia pastoris cells according to claim 3, characterized in that, The nucleotide sequence of the gene encoding the SEM1 factor is shown in SEQ ID NO:

1.

5. A Pichia pastoris engineered strain with enhanced exogenous protein expression and secretion capabilities, characterized in that, The Pichia pastoris engineered strain with enhanced exogenous protein expression and secretion overexpresses the gene encoding ALG14 and expresses the gene encoding an exogenous protein, wherein the amino acid sequence of the ALG14 factor is shown in SEQ ID NO:7, and the exogenous protein is xylanase, cellulase, mannanase, glucose oxidase, phytase, or saccharifying enzyme.

6. The Pichia pastoris engineered strain with enhanced exogenous protein expression and secretion capacity according to claim 5, characterized in that, The Pichia pastoris engineered strain with enhanced exogenous protein expression and secretion also includes an overexpressed gene encoding the SEM1 factor, wherein the amino acid sequence of the SEM1 factor is shown in SEQ ID NO:5.

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