Method for producing recombinant proteins using PDI derived from Trichoderma sp. strains

Abstract

The present invention relates to a method for producing a recombinant protein using PDI derived from a Trichoderma strain. A mutant yeast containing a gene encoding PDI derived from a Trichoderma strain of the present invention highly expresses a target protein, and can be used to produce the target recombinant protein in high yield.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0175628 dated December 15, 2022, and all contents disclosed in the documents of this Korean patent application are incorporated herein by reference.

[0002] The present invention relates to a method for producing a recombinant protein using PDI derived from a Trichoderma strain. [Background technology]

[0003] The host cells commonly used for recombinant protein production include Escherichia coli (E. coli), yeast (S. cerevisiae), and animal CHO cells. Among these, yeast offers several advantages over E. coli. Unlike E. coli, yeast is a eukaryotic cell genetically identical to higher organisms. Its transcription and translation systems are similar to those of higher organisms, allowing for the removal of introns through splicing. It also possesses a secretory apparatus similar to the Golgi apparatus of higher organisms, allowing for the production and secretion of activated proteins through post-translational modification. Furthermore, compared to E. coli, yeast can secrete recombinant proteins extracellularly more efficiently, facilitating their isolation and purification. Unlike E. coli, yeast does not pose a problem with endotoxins, which must be removed when producing pharmaceutical proteins. Among these yeasts, S. cerevisiae is a representative example, and since its use in recombinant protein production was reported in 1981, numerous researchers have actively pursued research into this area. However, achieving high expression rates in S. cerevisiae requires multi-copy plasmids. However, when high-density cell culture is performed in large-scale fermenters, problems with the distribution, copy number, and stability of these plasmids often arise. Furthermore, because most glycolytic gene promoters are constitutive, using these promoters for protein expression can result in dominating cells that have lost the plasmid. Even with regulatable promoters, precise regulation is difficult. Recently, the facultative methylotrophic yeast Pichia pastoris has attracted attention as an alternative solution to these problems.Pichia pastoris is a methylotrophic yeast that allows foreign genes to be integrated into its chromosomal DNA. It also has the advantage of using AOX1 as a promoter for enzymes in the methanol utilization pathway, due to its extremely strong promoter.

[0004] Protein disulfide isomerase (PDI) is an enzyme that catalyzes the thiol:disulfide bond exchange reaction. It resides in the endoplasmic reticulum (ER) and transfers disulfide bonds to newly synthesized secretory proteins that enter the ER. PDI is an oxidoreductase that not only transfers disulfide bonds but also has the isomerization activity of cleaving incorrectly linked disulfide bonds and converting them into structurally stable disulfide bonds. If PDI does not function properly, substrates escape the ER and are degraded without being secreted into the Golgi apparatus or extracellular space. Therefore, the efficiency of secretory protein synthesis can vary depending on the level of PDI activity.

[0005] Therefore, attempts have been made to use PDIs in recombinant protein production. Examples include fusing a thermophilic fungal PDI to the amino terminus of a target protein to achieve extracellular secretion in Bacillus brevis (Kajino, T. et al. (2000) Appl. Environ. Microbiol. 66: 638-642), and co-expressing dsbC, a PDI, with a target protein in the oxidative cytoplasm of a mutant Escherichia coli (Bessette, P.H. et al. (1999) Proc. Natl. Acad. Sci. USA, 96: 13703-13708). However, extracellular secretion in Bacillus strains has the drawback of significantly low target protein yields due to issues such as the secretion of proteolytic enzymes by Bacillus strains, and co-expression of dsbC with a target protein in the cytoplasm is known to result in low expression rates. Summary of the Invention [Problem to be solved by the invention]

[0006] An example of this application is 1) producing a mutant yeast comprising simultaneously, sequentially or in reverse order, the following steps i) and ii): i) introducing a vector containing a gene encoding PDI (protein disulfide isomerase) derived from a strain of the genus Trichoderma into yeast; ii) introducing a vector containing a gene encoding a protein of interest into yeast; and 2) culturing the mutant yeast prepared in step 1); The present invention provides a method for producing a recombinant protein, comprising:

[0007] Another example of the present application provides a mutant yeast for producing a target protein, which contains a gene encoding protein disulfide isomerase (PDI) derived from a strain of the genus Trichoderma.

[0008] Another example of the present application provides a composition for producing a target protein in a mutant yeast, which comprises a gene encoding PDI (protein disulfide isomerase) derived from a Trichoderma (genus Trichoderma) strain or a recombinant vector containing the gene. [Means for solving the problem]

[0009] To achieve the above object, the present invention provides a mutant yeast strain transformed with a gene encoding protein disulfide isomerase (PDI) derived from a strain of the genus Trichoderma, and a method for producing a target protein using the same.

[0010] The present invention will now be described in more detail.

[0011] The present invention provides 1) producing a mutant yeast comprising simultaneously, sequentially or in reverse order, the following steps i) and ii): i) introducing a vector containing a gene encoding PDI (protein disulfide isomerase) derived from a strain of the genus Trichoderma into yeast; ii) introducing a vector containing a gene encoding a protein of interest into yeast; and 2) culturing the mutant yeast prepared in step 1); The present invention provides a method for producing a recombinant protein, comprising:

[0012] As used herein, the term "PDI (protein disulfide isomerase)" refers to an enzyme that catalyzes a thiol:disulfide bond exchange reaction, and may be used interchangeably with "protein disulfide isomerase."

[0013] The PDI in step i) of step 1) may be a PDI derived from a strain of the genus Trichoderma, preferably a PDI derived from a strain of Trichoderma reesei.

[0014] In one embodiment of the present invention, the PDI may comprise or consist of the amino acid sequence of SEQ ID NO: 14. Polypeptides having a partial deletion, modification, substitution, or addition of the amino acid sequence of SEQ ID NO: 14 are also within the scope of the present application if they have the same or corresponding enzymatic activity as PDI. Furthermore, regardless of the microbial origin, polypeptides having at least 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 96% or more, 98% or more, 99% or more, 99.5% or more, or 99.8% or more homology or identity to the amino acid sequence of SEQ ID NO: 14 and having the same or corresponding converting activity as PDI are also within the scope of the present application.

[0015] The yeast in steps i) and ii) of step 1) may be any one selected from the group consisting of strains of the genus Pichia, Candida, and Hansenula. In one example, the yeast may be Pichia pastoris, more specifically, the Pichia pastoris BG16 strain, but is not limited thereto.

[0016] The target protein in step ii) of step 1) may be a protein containing a disulfide bond within the protein, and may be a protein containing three or more disulfide bonds within the protein, but is not limited thereto.

[0017] In one example of the present invention, the target protein may be any one or more selected from the group consisting of transferrin, albumin, and insulin precursor, and the transferrin, albumin, and insulin precursor may be proteins derived from bovine, but are not limited thereto.

[0018] As used herein, the term "transferrin" may refer to serum transferrin, which may be used as one of the factors that replace serum (FBS, fetal bovine serum) in serum-free media (FBS-free media), etc. In one example, the transferrin may comprise or consist of the amino acid sequence of SEQ ID NO: 6, but is not limited thereto.

[0019] [SEQ ID NO: 6] TIFF2025528374000002.tif56168

[0020] As used herein, the term "albumin" may refer to serum albumin and may be used as one of the factors that replace serum in serum-free media, etc. In one example, the albumin may comprise or consist of the amino acid sequence of SEQ ID NO: 24, but is not limited thereto.

[0021] [SEQ ID NO: 24] TIFF2025528374000003.tif44168

[0022] As used herein, the term "insulin precursor" may refer to one of factors that replace serum in serum-free media, etc. In one example, the insulin precursor may comprise or consist of the amino acid sequence of SEQ ID NO: 30, but is not limited thereto.

[0023] [SEQ ID NO: 30] Spacer sequences are underlined TIFF2025528374000004.tif7168

[0024] As used herein, the term "vector" refers to a DNA construct containing the base sequence of a polynucleotide encoding a PDI derived from a Trichoderma strain or a polynucleotide encoding a target protein operably linked to an appropriate expression control region (or expression control sequence) to enable expression of the polynucleotide in a suitable host. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating the termination of transcription and translation. After being transformed into a suitable host cell, a vector can replicate and function independently of the host genome or can be integrated into the genome itself.

[0025] The vectors used in this application are not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, and pPICZαA vectors can be used.

[0026] For example, a polynucleotide encoding a Trichoderma strain-derived PDI or a target protein can be inserted into a chromosome using a vector for intracellular chromosome insertion. The polynucleotide can be inserted into a chromosome by any method known in the art, including, but not limited to, homologous recombination. A selection marker for verifying the presence or absence of the chromosomal insertion can be additionally included. The selection marker is used to select cells transformed with the vector, i.e., to verify the presence or absence of the insertion of the target nucleic acid molecule. Markers that confer selectable phenotypes, such as drug resistance, auxotrophy, resistance to cytotoxic drugs, or expression of a surface polypeptide, can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes, allowing for the selection of transformed cells.

[0027] As used herein, the term "transformation" refers to the introduction of a vector containing a specific polynucleotide into a host cell or microorganism, thereby enabling the expression of a polypeptide encoded by the polynucleotide in the host cell. A transformed polynucleotide can include any polynucleotide that can be expressed in the host cell, regardless of whether it is located intrachromosomally or extrachromosomally. The polynucleotide can also include DNA and / or RNA encoding a target polypeptide. The polynucleotide can be introduced in any form that can be introduced and expressed in the host cell. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression. The expression cassette typically contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette can be in the form of an autonomously replicating expression vector. The polynucleotide can also be introduced into the host cell in its own form and operably linked to sequences necessary for expression in the host cell, but is not limited thereto.

[0028] Furthermore, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the PDI derived from a Trichoderma strain of the present application or a target protein.

[0029] As used herein, the term "culturing" refers to growing the mutant yeast of the present application under appropriately controlled environmental conditions. The culturing process of the present application may be carried out using an appropriate medium and culture conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing may be, but is not limited to, a batch, continuous, and / or fed-batch culture.

[0030] As used herein, the term "culture medium" refers to a mixture of nutrients, primarily those required for culturing the mutant yeast of the present application, that provides nutrients and growth factors, including water, essential for survival and growth. Specifically, the medium and other culture conditions used to culture the mutant yeast of the present application can be any medium commonly used for culturing microorganisms without any particular limitations. However, the mutant yeast of the present application can be cultured under aerobic conditions in a conventional medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, while adjusting the temperature, pH, and other parameters. Specifically, culture media for yeast can be found in literature such as "Yeast," Current Protocols in Molecular Biology, 2017, and "Pichia Protocols," Methods in Molecular Biology, 2007.

[0031] Examples of carbon sources include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; amino acids such as glutamic acid, methionine, and lysine; and methanol or ethanol. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid can also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) can be used. A variety of other carbon sources can be used in appropriate amounts without limitation. These carbon sources can be used alone or in combination of two or more types, and are not limited thereto.

[0032] The nitrogen source may be an inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; or an organic nitrogen source such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more, and are not limited thereto.

[0033] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or a corresponding sodium-containing salt. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and other compounds, including amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the medium in a batch or continuous manner. However, they are not limited thereto.

[0034] During the cultivation of the mutant yeast of the present application, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the medium in an appropriate manner. Also, during cultivation, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, oxygen or an oxygen-containing gas can be injected into the medium to maintain an aerobic state, or nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection or to maintain anaerobic and microaerobic states, but this is not limiting.

[0035] In the present invention, the culture temperature during the culture step can be maintained at 20 to 45°C, specifically 25 to 40°C, and the culture can be performed for about 10 to 160 hours, but is not limited thereto.

[0036] The recombinant protein produced by the culture of the present invention can be secreted into the medium or remain intracellularly.

[0037] The method for producing a recombinant protein of the present invention may additionally include a step of producing the mutant yeast of the present invention, a step of preparing a medium for culturing the mutant yeast, or a combination thereof (in any order), for example, before the culturing step.

[0038] The recombinant protein production method of the present invention may further include a step of recovering the recombinant protein from the culture medium (the medium in which the culture was performed) or the mutant yeast. The recovery step may be additionally performed after the culturing step.

[0039] The recovery may involve collecting the target protein using an appropriate method known in the art during the culture of the mutant yeast of the present invention, such as a batch, continuous, or fed-batch culture method. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination of these methods may be used. The target protein can be recovered from the medium or mutant yeast using an appropriate method known in the art.

[0040] Furthermore, the recombinant protein production method of the present invention may additionally include a purification step. The purification may be carried out using an appropriate method known in the art. For example, when the recombinant protein production method of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be carried out chronologically (or sequentially) in any order, simultaneously, or integrated into one step, but are not limited thereto.

[0041] The present invention also provides a mutant yeast for producing a target protein, which comprises a gene encoding PDI (protein disulfide isomerase) derived from a strain of the genus Trichoderma.

[0042] The PDI derived from a strain of the genus Trichoderma, the target protein, and the yeast are as described above.

[0043] The mutant yeast may be one in which a gene encoding a PDI derived from a Trichoderma strain has been inserted or incorporated into the yeast genome, or one in which the gene encoding a PDI derived from a Trichoderma strain is present outside the yeast genome.

[0044] The mutant yeast may be one into which a gene encoding a PDI derived from a heterologous Trichoderma strain has been introduced, and which exhibits increased productivity of a target protein compared to yeast to which the gene has not been introduced.

[0045] For example, the mutant yeast with increased productivity of the target protein may have an increased target protein productivity of about 1% or more, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 26% or more, about 27% or more, about 28% or more, about 29% or more, about 30% or more, about 31% or more, about 32% or more, about 33% or more, about 34% or more, about 35% or more, about 36% or more, about 37% or more, about 38% or more, about 39% or more, about 40% or more, about 41% or more, about 42% or more, about 43% or more, about 44% or more, about 45% or more, about 46% or more, about 47% or more, about 48% or more, about 49% or more, about 50% or more, about 51% or more, about 52% or more, about 53% or more, about 54% or more, about 55% or more, about 56% or more, about 57% or more, about 58% or more, about 59% or more, about 60% or more, about 61% or more, about 62% or more, about 63% or more, about 64% or more, about 65% or more, about 66% or more, about 67% or more, about 68% or more, about 69% or more, about 70% or more, about 71% or more, about 72% or more, about 73% or more, about 74% or more, about 75% or more, about 76% or more, about 77% or more, about 78% or more, about 79% or more, about 80% or more, about 81 The increase may be 36% or more, about 37% or more, about 38% or more, about 39% or more, about 40% or more, about 41% or more, about 42% or more, about 43% or more, about 44% or more, about 45% or more, about 46% or more, about 47% or more, about 48% or more, about 49% or more, or about 50% or more (there is no particular limitation on the upper limit, and it may be, for example, about 200% or less, about 150% or less, about 100% or less, or about 50% or less).

[0046] In other examples, the mutant yeast with increased productivity may have a target protein productivity (or production capacity or production amount) increased by about 1.1-fold or more, about 1.12-fold or more, about 1.13-fold or more, 1.15-fold or more, 1.16-fold or more, 1.17-fold or more, 1.18-fold or more, 1.19-fold or more, about 1.2-fold or more, 1.25-fold or more, about 1.3-fold or more, about 1.4-fold or more, or about 1.5-fold or more (there is no particular upper limit, and it may be, for example, about 10-fold or less, about 5-fold or less, about 3-fold or less, or about 2-fold or less) compared to the parent strain or unmodified microorganism before mutation, but is not limited thereto. The term "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​in a range that is equal to or similar to the numerical value following the term "about," but is not limited thereto.

[0047] As used herein, the term "non-transformed microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, and may refer to a wild-type or naturally occurring strain itself, or a strain before its characteristics are altered by genetic mutation due to natural or artificial factors. For example, the non-transformed microorganism may refer to a strain into which a gene encoding a PDI derived from a Trichoderma strain described herein has not been introduced or before it has been introduced. The term "non-transformed microorganism" may be interchangeable with "pre-transformed strain," "pre-transformed microorganism," "non-mutated strain," "non-transformed strain," "non-mutated microorganism," or "reference microorganism." For example, the "non-transformed microorganism" may be a Pichia pastoris strain, and more specifically, may be a Pichia pastoris BG16 strain, but is not limited thereto.

[0048] The present invention also provides a composition for producing a target protein in a mutant yeast, which comprises a gene encoding PDI (protein disulfide isomerase) derived from a strain of the genus Trichoderma or a recombinant vector containing the gene.

[0049] The PDI derived from a strain of the genus Trichoderma, the target protein, and the yeast are as described above.

[0050] In one example, the production composition may further contain any suitable excipient commonly used in compositions for producing a target protein, and such excipients may be, for example, but are not limited to, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, or an isotonic agent.

[0051] The present invention also provides a method for increasing productivity of a target protein in a microorganism or a method for imparting the ability to produce a target protein to a microorganism, the method comprising introducing (e.g., transforming) a polynucleotide encoding PDI (protein disulfide isomerase) derived from a Trichoderma strain and / or a recombinant vector containing the polynucleotide into the microorganism. [Effects of the Invention]

[0052] The mutant yeast of the present invention containing a gene encoding PDI derived from a strain of the genus Trichoderma expresses a target protein at a high level, and can therefore be used to produce a target recombinant protein in high yield. [Brief explanation of the drawings]

[0053] [Figure 1] 1 is a diagram showing a schematic diagram of the pPICZFRT recombinant plasmid. [Figure 2] 1 is a diagram showing a schematic diagram of the pPICZFRT-bTRANSFERRIN(N495Q) recombinant plasmid. [Figure 3] 1 shows the results of SDS-PAGE for comparing the transferrin expression levels in culture supernatants of Pichia pastoris PDI-introduced strain (CF04-1024), Ogataea angusta PDI-introduced strain (CF04-1025), and Trichoderma ressei PDI-introduced strain (CF04-1026). [Figure 4]1 is a diagram showing a schematic diagram of the pPICZFRT-PDI(TR) recombinant plasmid. [Figure 5] 1 shows the results of SDS-PAGE comparing the expression levels of albumin in the culture supernatants of Pichia pastoris BG16 (CF04-1005) and CF04-1035 (CF04-1038) strains, respectively, in which a gene encoding bovine albumin was introduced. [Figure 6] FIG. 1 shows the results of SDS-PAGE to compare the expression levels of bovine insulin precursor in the culture supernatants of Pichia pastoris BG16 (CF04-1017) and CF04-1035 (CF04-1040) strains, respectively, in which a gene encoding the bovine insulin precursor was introduced. DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention will be described in more detail below with reference to examples. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

[0055] Example 1. Preparation of pPICZFRT plasmid

[0056] A vector for transformation of Pichia pastoris was prepared as follows.

[0057] DNA (SEQ ID NO: 1) consisting of an FRT site, restriction enzyme recognition sites for cloning, the LRA3 (L-rhamnonate dehydratase) promoter (expressed under rhamnose-inducing conditions), and Flp recombinase was synthesized and ligated to the 1341-3593 bp region of the pPICZαA vector (Invitrogen™, catalog number: V19520). The region of SEQ ID NO: 1 was amplified by PCR using the DNA of SEQ ID NO: 1 synthesized for this genetic manipulation as a template and primers with the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3. The 1341-3593 bp region of the pPICZαA vector was amplified by PCR using the pPICZαA vector DNA as a template and primers with the nucleotide sequences of SEQ ID NO: 4 and SEQ ID NO: 5. PCR reactions were performed using Pfu-X DNA Polymerase (Solg™, catalog number: SPX16-R500) according to the manufacturer's protocol. The two amplified fragments were cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL. 6 NO. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) to obtain a recombinant plasmid named pPICZFRT (see Figure 1).

[0058] Example 2. Preparation of Pichia pastoris strain expressing non-glycosylated bovine transferrin

[0059] The amino acid sequence of bovine transferrin was determined from NCBI reference sequence ID: NP_803450.2, excluding the signal sequence, and consisted of residues 19 to 704. To remove glycosylation from this sequence, N (asparagine) at residue 495 was replaced with Q (glutamine), resulting in the non-glycosylated form. The amino acid sequence of non-glycosylated bovine transferrin is shown in SEQ ID NO: 6.

[0060] The DNA codons were optimized to express a protein having the amino acid sequence of SEQ ID NO: 6 in Pichia pastoris. The DNA codon-optimized base sequence is shown in SEQ ID NO: 7.

[0061] A DNA fragment was constructed by gene synthesis using the AOX1 promoter for methanol-inducible expression, followed by an alpha-mating factor sequence for secretory expression, and an AOX1 transcription terminator sequence. The resulting fragment was amplified by PCR using primers with SEQ ID NOs: 8 and 9 and Pfu-X DNA polymerase. pPICZFRT was then digested with KpnI-HF (NEB, Catalog No. R3142) and NotI-HF (NEB, Catalog No. R3189). The resulting two fragments were cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, Vol. 6, No. 5, May 2009, NEBuilder HiFi DNA Assembly Master Mix) to obtain a recombinant plasmid designated pPICZFRT-bTRANSFERRIN(N495Q) (see Figure 2).

[0062] To transform a Pichia pastoris strain with the pPICZFRT-bTRANSFERRIN(N495Q) vector, the AOX1 promoter site was linearized using PmeI (NEB, catalog number: R0560S). The linearized vector was then purified using the Expin™ PCR SV kit (GeneAll, catalog number: 103-102). The Pichia pastoris strain used was PPS-9016 [Pichia pastoris BG16 (pep4△, prb1△, protease deficient)] purchased from ATUM. The linearized pPICZFRT-bTRANSFERRIN(N495Q) vector was transformed into Pichia pastoris BG16 by electroporation according to the ATUM manual. Transformants were selected for resistance to zeocin (250 μg / mL) antibiotic (Gibco®, catalog number: R25001) on YPDS agar medium (yeast extract 10 g / L, peptone 20 g / L, sorbitol 182.2 g / L, bacto agar 20 g / L). Integration into the AOX1 promoter site in the genome was confirmed using a forward primer (SEQ ID NO: 10) that binds to the 21-bp portion of the AOX1 promoter in the genome and a reverse primer (SEQ ID NO: 11) that binds to the Rhamnose promoter of the vector. PCR reactions were performed using the Phire Plant Direct PCR Kit (Thermo Scientific®, catalog number: F130WH) according to the manufacturer's protocol.

[0063] Clones whose transformation was confirmed by PCR were inoculated into 1 mL of YP (yeast extract 10 g / L, peptone 20 g / L) + 1% rhamnose liquid medium and cultured at 30°C and 200 rpm for 24 hours. To select clones in which the FRT region had been deleted due to the recognition of the FRT site by Flp recombinase induced by rhamnose, the clones were streaked onto YPD agar medium (yeast extract 10 g / L, peptone 20 g / L, bacto agar 20 g / L) and cultured at 30°C for 72 hours. Single colonies were streaked onto YPD agar medium and YPDS + zeocin agar medium, respectively, to select colonies that had lost zeocin resistance due to the deletion of the FRT region. The selected strain was designated CF04-1023.

[0064] Example 3. Construction of Pichia pastoris strains incorporating PDI orthologues from various strains and confirmation of bovine transferrin expression

[0065] Example 3-1. Construction of vectors for introducing PDIs from various strains

[0066] To confirm the effect of introducing PDIs derived from various strains into the bovine transferrin-expressing strain CF04-1023 prepared in Example 2 on bovine transferrin expression, vectors incorporating PDIs derived from various strains were prepared as follows.

[0067] Based on NCBI BlastP of the Pichia pastoris PDI amino acid sequence (SEQ ID NO: 12), PDI from Ogataea angusta (SEQ ID NO: 13), which has 54.63% homology to the amino acid sequence of SEQ ID NO: 12, and PDI from Trichoderma reesei (SEQ ID NO: 14), which has 44.20% homology, were selected as test candidates.

[0068] Specifically, for expression in Pichia pastoris, a gene fragment of Pichia pastoris PDI was isolated from gDNA by PCR using primers having the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 16. The sequences of Ogataea angusta PDI and Trichoderma reesei PDI are SEQ ID NO: 17 and SEQ ID NO: 18. Each sequence was synthesized by adding 20 bp of sequence complementary to the pPICZαA vector (Invitrogen®, catalog number: V19020) sequence before and after the vector for cloning. Using the synthesized DNA as a template, the respective gene fragments were isolated by PCR using primers having the nucleotide sequences of SEQ ID NO: 19 and SEQ ID NO: 20 and Pfu-X DNA Polymerase. Each gene fragment was linearized with EcoRI-HF (NEB, catalog number R3101S) and cloned into pPICZA vector using Gibson assembly (DG Gibson et al., NATURE METHODS, VOL. 6 NO. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) to obtain recombinant plasmids. The vector containing Pichia pastoris PDI was named pPICZA-PDI(PP), the vector containing Ogataea angusta PDI was named pPICZA-PDI(OA), and the vector containing Trichoderma reesei PDI was named pPCIZA-PDI(TR).

[0069] Example 3-2. Preparation of Pichia pastoris strains incorporating PDIs from various strains

[0070] The bovine transferrin-expressing Pichia pastoris BG16 strain CF04-1023 prepared in Example 2 was transformed with the pPICZA-PDI(PP), pPICZA-PDI(OA), and pPICZA-PDI(TR) vectors prepared in Example 3-1 as described below to prepare strains into which PDIs derived from various strains were introduced.

[0071] Specifically, the AOX1 promoter region of each vector was linearized using PmeI (NEB, catalog number: R0560S). The linearized vectors were then purified using the Expin™ PCR SV kit (GeneAll, catalog number: 103-102). Each linearized vector was electroporated into the CF04-1023 strain according to the ATUM manual, and transformants were selected on YPDS agar medium for resistance to the antibiotic zeocin (250 μg / mL). Integration into the AOX1 promoter region within the genome was confirmed using a forward primer (SEQ ID NO: 10) that binds to the 21-bp portion of the AOX1 promoter in the genome and a reverse primer (SEQ ID NO: 21) that binds to the TEF1 promoter of the vector. PCR reactions were performed using the Phire Plant Direct PCR Kit according to the manufacturer's protocol. Of the selected strains, the Pichia pastoris PDI-introduced strain was named CF04-1024, the Ogataea angusta PDI-introduced strain was named CF04-1025, and the Trichoderma ressei PDI-introduced strain was named CF04-1026.

[0072] Example 3-3. Confirmation of bovine transferrin expression levels according to various strain-derived PDIs introduced into Pichia pastoris strains

[0073] To confirm the expression level of bovine transferrin for each of the various strain-derived PDIs introduced into Pichia pastoris strains, the four strains (CF04-1023, 1024, 1025, and 1026) prepared in Example 3-2 were cultured and the expression of bovine transferrin was confirmed.

[0074] The strain was cultured in 25 ml of BMMY liquid medium (1% yeast extract, 2% peptone, 13.4 g / l yeast nitrogen base (without amino acids), 100 mM potassium phosphate (pH 6.0), 0.004 mg / l biotin, 1% methanol) in a 250 ml baffled Erlenmeyer flask at 30°C and 200 rpm for 96 hours. Additional 1% methanol was added every 24 hours to induce sustained expression of the protein under the control of the AOX1 promoter. After fermentation, the presence or absence of expression was confirmed by analyzing the culture supernatant. The culture supernatant, after sedimentation of yeast cells by centrifugation, was separated on a 4-20% Tris-glycine polyacrylamide gel by SDS-PAGE (Laemmli, 1970) and stained using DIRECTBLUE™ gel staining solution (SCGBIOMAX, catalog number: BDS-1000). The transferrin band density on the gel was analyzed using a calibrated densitometer (BIO-RAD, GS-900). To further confirm whether the band of the specified size was transferrin, it was analyzed by LC-MS / MS analysis using in-gel digestion (Rosenfeld, 1992). The transferrin expression SDS-PAGE results are shown in Figure 3, and the comparison of transferrin expression levels is shown in Table 1 below.

[0075] [Table 1]

[0076] As a result, as shown in Figure 3 and Table 1, the strain without PDI (CF04-1023) did not express bovine transferrin, whereas only the strain with PDI added expressed bovine transferrin. Furthermore, compared to the strains with Ogataea angusta PDI and Pichia pastoris PDI, the strain with Trichoderma reesei PDI, CF04-1026, showed the highest bovine transferrin expression level, approximately 1.3-fold higher than the strain with Pichia pastoris PDI added.

[0077] Example 4. Preparation of Trichoderma reesei PDI-expressing strain

[0078] In Example 3-3, it was confirmed that the Pichia pastoris strain expressing non-glycosylated bovine transferrin, into which Trichoderma reesei PDI had been introduced, had the highest bovine transferrin expression level. Therefore, a Pichia pastoris strain expressing Trichoderma reesei PDI was prepared as follows.

[0079] Using pPCIZA-PDI(TR) as a template and primers with the nucleotide sequences of SEQ ID NO: 22 and SEQ ID NO: 23, a DNA fragment consisting of the AOX1 promoter, Trichoderma reesei PDI, and AOX1 terminator was obtained by PCR using Pfu-X DNA Polymerase. pPICZFRT was digested with KpnI-HF and NotI-HF, and the two fragments were cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL. 6 NO. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) to obtain a recombinant plasmid named pPICZFRT-PDI(TR) (see Figure 4). The pPICZFRT-PDI(TR) vector was introduced into Pichia pastoris BG16 using the same method as described in Example 2. A clone in which Trichoderma reesei PDI was integrated into the genome of Pichia pastoris BG16 and zeocin resistance was lost through excision of the FRT site was selected. This strain was designated CF04-1035.

[0080] Example 5. Confirmation of bovine albumin expression in Trichoderma reesei PDI-expressing strain

[0081] To confirm whether the strain is superior in expressing other proteins besides bovine transferrin, the bovine albumin gene was introduced into the Trichoderma reesei PDI-expressing strain (CF04-1035) prepared in Example 4 and the Pichia pastoris BG16 strain, and the expression levels were compared. The amino acid sequence of bovine albumin is SEQ ID NO: 24, which uses the sequence from 25 to 607 of Uniprot ID: P02769, excluding the signal sequence.

[0082] To express and secrete bovine albumin protein (SEQ ID NO: 24), the DNA sequence encoding it was codon-optimized and synthesized by adding 20 bp of sequences complementary to the pPICZαA vector (Invitrogen™, catalog number: V19520) sequence before and after the vector for cloning. The resulting sequence is shown in SEQ ID NO: 25. Using the synthesized DNA as a template, PCR was performed with Pfu-X DNA Polymerase using primers with the nucleotide sequences of SEQ ID NO: 26 and SEQ ID NO: 27 to obtain a gene fragment. Then, PCR was performed with primers with the nucleotide sequences of SEQ ID NO: 28 and SEQ ID NO: 29 to obtain a gene fragment for use as a vector using the pPICZαA vector as a template. The two obtained gene fragments were cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL. 6 NO. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) to obtain a recombinant plasmid. The obtained vector was named pPCIZαA-bALBUMIN. The pPCIZαA-bALBUMIN vector was introduced into the BG16 strain and the CF04-1035 strain, and the vector was confirmed using the same method as described in Example 2. The strain in which a gene encoding bovine albumin was introduced into the Pichia pastoris BG16 strain was designated CF04-1005, and the strain in which a gene encoding bovine albumin was introduced into CF04-1035 was designated CF04-1038.

[0083] The two strains were cultured and the expression levels of bovine albumin were compared in the same manner as described in Example 3-3. The results are shown in FIG. 5 and Table 2 below.

[0084] [Table 2]

[0085] As a result, as shown in Figure 5 and Table 2, it was confirmed that the bovine albumin expression level of the CF04-1038 strain, which expresses Trichoderma reesei PDI, was approximately 1.4 times higher than that of the CF04-1005 strain, which does not have Trichoderma reesei PDI introduced.

[0086] Example 6: Confirmation of bovine insulin precursor expression in Trichoderma reesei PDI-expressing strain

[0087] To confirm whether Trichoderma reesei PDI-expressing strains are superior in expressing bovine insulin precursors, we introduced a gene encoding bovine insulin precursor into the Trichoderma reesei PDI-expressing strain (CF04-1035) prepared in Example 4 and the Pichia pastoris BG16 strain, and compared the expression levels. The amino acid sequence of bovine insulin precursor is Uniprot ID: I7CLV3, and uses the sequence 1-30 corresponding to the B chain of insulin and the sequence 61-81 corresponding to the A chain. An EPK sequence was added before the B chain and an AAK sequence was added between the B chain and the A chain (Thomas Kjeldsen, 2002, JBC vol. 277, issue 21). The sequence is shown in SEQ ID NO: 30.

[0088] To express and secrete bovine insulin precursor protein (SEQ ID NO: 30), the DNA sequence encoding this protein was codon-optimized and synthesized with 20 bp of complementary sequences to the pPICZαA vector sequence added before and after for cloning. The resulting sequence is shown in SEQ ID NO: 31. Using the synthesized DNA as a template, a gene fragment was obtained by PCR using primers with the nucleotide sequences of SEQ ID NO: 26 and SEQ ID NO: 27. Then, using the pPICZαA vector as a template, a gene fragment for use as a vector was obtained by PCR using primers with the nucleotide sequences of SEQ ID NO: 28 and SEQ ID NO: 29. The two obtained gene fragments were cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL. 6 NO. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) to obtain a recombinant plasmid. The obtained vector was named pPCIZαA-bPROINSULIN. The pPCIZαA-bPROINSULIN vector was introduced into the BG16 strain and the CF04-1035 strain, and the vector was confirmed using the same method as described in Example 2. The Pichia pastoris BG16 strain into which a gene encoding a bovine insulin precursor was introduced was designated CF04-1017, and the CF04-1035 strain into which it was introduced was designated CF04-1040.

[0089] The two strains were cultured and the expression levels of bovine insulin precursor were compared. The experiment was conducted in the same manner as in Example 3-3, except that 10-20% Tris-tricine polyacrylamide gel was used instead of 4-20% Tris-glycine polyacrylamide gel. The results are shown in Figure 6 and Table 3 below.

[0090] [Table 3]

[0091] As a result, as shown in Figure 6 and Table 3, it was confirmed that the expression level of bovine insulin precursor in the CF04-1040 strain, which expresses Trichoderma reesei PDI, was approximately 1.5 times higher than that in the CF04-1017 strain, which does not have Trichoderma reesei PDI introduced.

Claims

1. 1) producing a mutant yeast comprising simultaneously, sequentially or in reverse order, the following steps i) and ii): i) introducing a vector containing a gene encoding PDI (protein disulfide isomerase) derived from a strain of the genus Trichoderma into yeast; ii) introducing a vector containing a gene encoding a protein of interest into yeast; and 2) culturing the mutant yeast prepared in step 1); A method for producing a recombinant protein comprising:

2. 2. The method for producing a recombinant protein according to claim 1, wherein the Trichoderma strain in step i) of step 1) is Trichoderma reesei.

3. 2. The method of claim 1, wherein the PDI (protein disulfide isomerase) derived from a Trichoderma strain in step 1) i) has the amino acid sequence of SEQ ID NO:

14.

4. 2. The method of claim 1, wherein the yeast is any one selected from the group consisting of strains of the genus Pichia, Candida, and Hansenula.

5. 2. The method for producing a recombinant protein according to claim 1, wherein the yeast is a Pichia pastoris strain.

6. 2. The method of claim 1, wherein the target protein in step 1) ii) is a protein containing three or more disulfide bonds.

7. 7. The method of claim 6, wherein the target protein is at least one selected from the group consisting of transferrin, albumin, and insulin precursor.

8. 8. The method for producing a recombinant protein according to claim 7, wherein the transferrin, albumin and insulin precursor are derived from bovine.

9. A mutant yeast for producing a target protein, which contains a gene encoding PDI (protein disulfide isomerase) derived from a strain of the genus Trichoderma.

10. The mutant yeast according to claim 9 , wherein the gene encoding the PDI derived from a Trichoderma strain is inserted into the yeast genome.

11. 10. The mutant yeast of claim 9, wherein the strain of the genus Trichoderma is Trichoderma reesei.

12. The mutant yeast according to claim 9 , wherein the mutant yeast is any one selected from the group consisting of the genus Pichia, the genus Candida, and the genus Hansenula.

13. 10. The mutant yeast of claim 9, wherein the mutant yeast is a Pichia pastoris strain.

14. The mutant yeast according to claim 9 , wherein the target protein is a protein containing three or more disulfide bonds.

15. The mutant yeast according to claim 9 , wherein the target protein is at least one selected from the group consisting of transferrin, albumin, and an insulin precursor.

16. The mutant yeast of claim 15 , wherein the transferrin, albumin, and insulin precursor are derived from bovine.

17. A composition for producing a target protein in a mutant yeast, comprising a gene encoding PDI (protein disulfide isomerase) derived from a strain of the genus Trichoderma or a vector containing the same.

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