WO2013191369A1 - Hpgas1 gene-disrupted yeast strain and production method for recombinant protein using same - Google Patents

Hpgas1 gene-disrupted yeast strain and production method for recombinant protein using same Download PDF

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WO2013191369A1
WO2013191369A1 PCT/KR2013/003581 KR2013003581W WO2013191369A1 WO 2013191369 A1 WO2013191369 A1 WO 2013191369A1 KR 2013003581 W KR2013003581 W KR 2013003581W WO 2013191369 A1 WO2013191369 A1 WO 2013191369A1
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gene
strain
hpgasl
polymorpha
protein
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PCT/KR2013/003581
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French (fr)
Korean (ko)
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권오석
황동현
오두병
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한국생명공학연구원
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)

Definitions

  • Hp GA Gene Fragmenting Yeast Strain and Method of Production of Recombinant Protein Using the Same
  • the present invention relates to a mutant strain that breaks a novel gene to improve protein secretion ability and a foreign recombinant protein production method using the mutant strain.
  • Protein drug development began in the 1970s with the introduction of genetic recombination technology.
  • the recombinant protein pharmaceutical production technology which produces a large amount of useful proteins such as hormones, antibodies, vaccines, and biofunctional proteins in a microbial host cell and develops them into pharmaceuticals, utilizes representative microorganisms that utilize microorganisms as cell factories.
  • the demand for high-purity proteinaceous medicines for the treatment of intractable diseases is growing exponentially, and recombinant medicines using microbial expression systems capable of mass production at low cost Production technology development is expected to contribute greatly to the growth of the future pharmaceutical industry.
  • Early protein drugs were sometimes isolated from human tissue or blood, but due to serious problems such as viral infections, including HIV and hepatitis, and the potential for residual cancer-causing agents,
  • Recombinant protein production technology is a technology that produces high value-added medical proteins with limited production cost by producing genes derived from higher organisms from a variety of microorganisms through gene recombination technology developed relatively).
  • the demand for high-purity proteinaceous drugs is expected to soar due to the increase of diseases and the improvement of national medical standards. Therefore, there is a need for a technology development research capable of mass production of new functional recombinant protein at a relatively low cost using various microorganisms that are harmless to the human body.
  • methanol magnetization yeast Hansenula polymorphaV (3 ⁇ 4 £ / / 3 polymorphaV): It is possible to cultivate relatively high concentrations easily using methanol, a cheap raw material as a carbon source, and a strong promoter derived from several genes involved in metabolism. In addition, the foreign gene is transferred to the host cell's chromosomal DNA. Multicopy It can be integrated) even during high concentration cultivation : it has the advantage of keeping stable.
  • the present inventors have proposed a novel beta-1 that is critical for cell wall composition and permeability determination in order to further increase the production of protein secretion of the existing Hansenula polymorpho ( ⁇ e / / a polyworpha) strain, which is advantageous for the production of foreign recombinant proteins.
  • HpGASl gene having 3-glucanosyl transglycosy lase (beta-1, 3-glucanosyl transglycosy lase) activity was secured, it was confirmed that the foreign recombinant protein secretion ability increases when the gene is deleted, through the present invention
  • the present invention was completed by revealing that a single-deleted Hansenula polymorpha mutant strain having a single deletion of HpGASl gene can be usefully used as a resource for secretory production of a pharmaceutical recombinant protein.
  • Another object of the present invention to provide a method for producing a yeast strain with improved protein secretion ability.
  • Another object of the present invention is to provide a foreign recombinant protein production method.
  • Another object of the present invention is to provide a use of a Hanshenula polymorpho Ofe ⁇ ey / polymorpha) strain that is deficient in the HpGASl gene as set forth in SEQ ID NO: 1 to enhance protein secretion ability.
  • the present invention is HpGASl described in SEQ ID NO: 1 It provides a Hanshenula polymorpha 03 ⁇ 4 7se // a polyworpha) variant strain of Accession No. KCTC12220BP with improved gene secretion ability.
  • the present invention also provides a method for producing a yeast strain with improved protein secretion ability.
  • the present invention also provides a foreign recombinant protein production method.
  • the present invention provides a use of a Hansennura polymorpha 03 ⁇ 472s 3 ⁇ 4 // a poly orpha) mutant strain lacking the HpGASl gene as set forth in SEQ ID NO: 1 for improving protein secretion ability.
  • Figure 2 shows the results of searching for homology to the sequence of the HpGAS2 gene described in SEQ ID NO: 2 using BLAST of NCBI.
  • Hydrophobic region Hydrophobic region
  • Figure 4 is a diagram showing the manufacturing process of the ⁇ gene disruption cassette.
  • FIG. 5 is a diagram illustrating a manufacturing process of the HpGASl gene disruption cassette.
  • FIG. 6 is a diagram showing the manufacturing process of the ⁇ 5 gene disruption cassette.
  • FIG. 8 is a photograph showing the cell morphological features of HpGASl and HpGAS2 single deletion mutants.
  • A 2% glucose complex medium (37 0 C); B: 2% glucose complex medium (42 ° C.);
  • Hanshenula polymorpha strain (polymorpha DLl AHpGASl ⁇ 3 ⁇ 43 ⁇ 45 / pDLGUK-HpGASl) lacking the HpGASl gene transformed with HpGASl expression vector (pDLGUK-HpGASl);
  • Hansenula polymorpha strain lacking the HpGASl gene transformed with HpGAS2 expression vector P DLGUK-HpGAS2 (.polymorpha DLl AHpGASl ⁇ j pDLGUK-HpGAS2);
  • Hansenula polymorpha DL1 wild type strain (.polymorpha DLl);
  • FIG. 11 is a photograph comparing Glucose oxidase (GOD) secretion in HpGASl and HpGAS2 single deletion strains:
  • HpGAS-deficient GOD producing strain [polymorpha AffpGAS pOLUOI-GODiR)].
  • Figure 13 is a photograph comparing human serum albumin (HSA) secretion in HpGASl and HpGAS2 single deletion strains:
  • HSA producing strains deficient in HpGAS (.polymorpha G3T8 AHpGAS2).
  • 14 is a diagram showing a process of H PGASl gene disrupted strains and H P UR / deletion. '
  • Figure 15 is a photograph confirming the physiological characteristics of strains transformed HpGASl and 3 ⁇ 4? Fi45 gene expression vector in 5. cerevisiae BY4741 lacking the ScGASl gene:
  • A 2% glucose complex medium
  • B Sodium Dodecyl Sulfate (SDS) ⁇ 2% galactose and 1% raffinose complex medium
  • C Congo Red (CR)-2% galactose and 1% raffinose minimal medium
  • Saccharomyces cerevisiae strain (5. cerevisiae BY4741 15c ⁇ 5i / YEp352ScGAPDHpt -HpGAS2) that lacks the 5 45? Gene transformed with ⁇ A expression vector (YEp352ScGAPDHpt-HpGAS2); and
  • Saccharomyces cerevisiae strain S. cerevisiae BY4741 l & i3 ⁇ 45i / YEp352ScGAPDHpt
  • Saccharomyces cerevisiae strain S. cerevisiae BY4741 l & i3 ⁇ 45i / YEp352ScGAPDHpt
  • YEp352ScGAPDHpt empty site
  • the present invention provides Hansenula polymorpha ( ⁇ ;? se // a polymorpha) mutant strain of Accession No. KCTC12220BP with improved protein secretion ability, wherein the HpGASl gene described in SEQ ID NO: 1 is deleted.
  • the inventors obtained HpGAS gene candidates for translating proteins having amino acid sequences similar to those of other yeast Gasl proteins in the Hanshenula polymorpha DL1 genomic information database.
  • two Hansenula polymorpha genes ( ⁇ ⁇ 5 ⁇ HpGAS2) o] were analyzed to translate Gasl proteins of other yeasts and proteins having high amino acid sequence homology (see FIGS. 1 and 2).
  • HpGASl gene of a novel sequence that translates a protein having higher homology with the amino acid sequence of ScGasl protein of Saccharomyces cerevisiae The amino acid sequence and the active domain conserved in several Gasl proteins exist in the amino acid sequence of the translating protein It was confirmed that (see Figure 3).
  • HpGASl ⁇ ⁇ ⁇ 5 gene was disrupted first, the Hansenula polymorpha U A3 gene was disrupted with a disruption cassette, followed by homologous recombination using HpGASl and H P GAS2 gene disruption cassettes. The HpGASl and H P GAS2 genes of the morpha strains were respectively disrupted.
  • the ⁇ gene disruption cassette was prepared using an N-terminal fragment of the HpURA3 gene, a C-terminal fragment of the HpURA3 gene, and a Zeocin resistance gene (Zeo R ) cassette. It is preferably a crushed cassette, but is not limited thereto.
  • the HpGASl gene disruption cassette comprises an N-terminal fragment of the HpGASl gene, a C-terminal fragment of the HpGASl gene, and 7ac -y3 ⁇ 4? N-terminal fragment of cassette, 1 acZ ⁇ HpURA3- 1 acZ? It is preferable to use a shredding cassette produced using a sheet C-terminal fragment, but is not limited thereto.
  • the inventors of the present invention in order to disrupt the ⁇ 3 gene to distinguish gene disruption, the N terminal segment of the H P URA3 gene, the C terminal segment of the HpUM3 gene and Zeocin resistance gene (Zeo R ) cassette Using the «fragment cassette was transformed to Hanshenula polymorpha strain and then the corresponding gene on the chromosome was disrupted by homologous recombination method (see Fig. 4).
  • the N-terminal fragment of the HpGASl gene, the C-terminal fragment of the HpGASl gene, lacZ-HpUM3-lacZ 7 ⁇ hent ⁇ N-terminus fragment, and 1 acZ-HpURA3- 1 acZ? ⁇ ⁇ terminal fragment were prepared in the fragmented strain.
  • One disrupted cassette was transformed to prepare a strain in which the HpGASl gene was crushed (see FIG. 5), and it was confirmed that the protein secretion ability of the strain in which the ⁇ i S gene was crushed was improved.
  • the recombinant yeast strain is Saccharomyces (5ac 3 ⁇ 4 / 3 ⁇ 4z ⁇ es), Kluberomyces Uduyveroinyces, Picky ⁇ iPichia, Hanshenula 03 ⁇ 4 / 7se //) or It is preferably any one selected from the group consisting of Candida genus, and more preferably, but not limited to Hanshenula polymorpha strain.
  • the foreign recombinant production method preferably further comprises the step of culturing the mutant strain using a bioreactor, but is not limited thereto.
  • the inventors have introduced a vector comprising a gene encoding glucose oxidase (GOD) or human serum albumin (HSA) to introduce HpGASl or GOD-producing strains and HSA-producing strains that produce foreign recombinant proteins.
  • GOD glucose oxidase
  • HSA human serum albumin
  • the Hanshenula polymorpha strain in which the HpGASl gene of the present invention is singly disrupted can be usefully used as a resource for the production of foreign recombinant proteins.
  • the present invention provides a use of a Hansennura polymorpha (? / 7 «? / 3 polymorpha) variant strain that lacks the HpGASl gene as set forth in SEQ ID NO: 1 to improve protein secretion ability.
  • HpGASl genes were identified through translation and search.
  • HpGAS gene candidates were obtained through a tblastn program for translating proteins having amino acid sequences similar to those of Gasl proteins.
  • the two highly homologous genes were compared and selected for homology between the other yeast Gasl protein and amino acid sequence and named HpGASl gene (SEQ ID NO: 1) and ⁇ ⁇ 45 gene (SEQ ID NO: 2) (Table 1).
  • the present inventors cloned the Zeocin resistance gene (Zeo R ) between URA3 gene segments with the URA3 disruption cassette to disrupt the ⁇ 3 gene of Hanshenula polymorpha strain and make it a nutritional requirement.
  • Zeo R Zeocin resistance gene
  • the Zeocin resistance gene (Zeo R ) cassette (1192 bp) was obtained by polymerase chain reaction using Zeo resistance ⁇ Forward (SEQ ID NO: 7) and Zeo resistance_Reverse (SEQ ID NO: 8) using the pPICZalphaA vector as a template.
  • the URA3 disruption cassette was prepared using the HpURA3 ⁇ ⁇ N-terminal fragment, Zeocin resistance gene (Zeo R ) cassette, and HpURA3 ⁇ C-terminal fragment prepared above by Fusion PCR. ⁇ 3 crush cassette was used to produce wild type Hanshenula polymorpha DLl), Glucose oxidase (GOD) recombinant strain [(? R /?
  • the present inventors prepared a GAS1 disruption cassette "to generate a disrupted strain lacking the HpGASl gene and introduced it into the mutant strain of Example ⁇ 2-1> in which the U 3 gene was disrupted.
  • ⁇ 45 _ / _ N_Forward (SEQ ID NO: 9) and v3 ⁇ 4o ⁇ 5_N_Reverse_UJF (SEQ ID NO: 9) were used as chromosomal DNA isolated from Hanshenula polymorpha DL1 strain. 10) was used to obtain HpGASl ⁇ ⁇ N-terminal fragment (537 bp), and ⁇ p5SlS ⁇ C- using ⁇ 5 _CJ rward— LUR (SEQ ID NO: 11) and ⁇ A _C— Reverse (SEQ ID NO: 12). Terminal fragments (524 bp) were obtained via polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • pLaclIR3 as a template (Kim et al., J Biol Chem. 281, 6261, 2006) using primer pairs Hp 1 acZ_URA3_N_Forward (SEQ ID NO: 24) and HplacZ_URA3_N_Reverse (SEQ ID NO: 25) for the expression of lacZ-H P URA3-lacZ cassette.
  • N-terminal fragments were obtained by HplacZ_URA3_C_Forward (SEQ ID NO: 26) and Hp lacZ_URA3_C—Reverse (SEQ ID NO: 27) to obtain C-terminal fragments of the lacZ-H P U A3-lacZ cassette by polymerase chain reaction.
  • HpGASlS ⁇ N-terminal fragment, N-terminal fragment of lacZ-HpURA3-lacZ cassette, were obtained by GAS1 disruption cassette _N-terminal fragment by fusion PCR, C-terminal fragment of lacZ-HpURA3-lacZ cassette, HpGASl ⁇ C-terminal Sections were obtained by GAS1 disruption cassette _C ⁇ terminal fragments by fusion PCR (Fig. 5).
  • the prepared GAS1 disrupted cassette ⁇ N-terminal fragment and the GAS1 disrupted cassette _C-terminal fragment were introduced into each of the strains of the URA3 gene fragmented in Example ⁇ 2-1> by homologous recombination, and then, in the SC-URA minimal medium.
  • a mutant strain in which the viable HpGASl gene was disrupted was obtained.
  • the inventors prepared a disruption cassette to introduce a mutant strain in which the UM3 gene of Example ⁇ 2-1> was disrupted to prepare a disrupted strain lacking the H P GAS2 gene.
  • HPGAS2 2 C-terminal fragment (524 bp) was obtained through polymerase chain reaction (PCR) using ⁇ ⁇ 5 ⁇ C_Forward_LU (SEQ ID NO: 15) and ⁇ fi! S2 _Reverse (SEQ ID NO: 16).
  • the N-terminal fragment of the lacZ—HpU A3-lacZ cassette was prepared using the primer pairs HplacZ_URA3_N_Forward (SEQ ID NO: 24) and HplacZ—URA3_N_Reverse (SEQ ID NO: 25).
  • C-terminal fragments of the lacZ-HpURA3-lacZ cassette were obtained by polymerase chain reaction using HplacZ_URA3_C_Forward (SEQ ID NO: 26) and HplacZ_URA3_C_Reverse (SEQ ID NO: 27).
  • HpGAS2 ⁇ ⁇ N ⁇ fragment and N ⁇ terminal fragment of lacZ-HpURA3-lacZ cassette were subjected to fusion PCR.
  • N-terminal fragments were obtained from the shredding cassette
  • H P GAS2 C-terminal fragments were obtained from the GS ⁇ crushing cassette_C-terminal fragments by fusion PCR (FIG. 6).
  • the resulting fragmented cassette ⁇ ⁇ -terminal fragment and the GAS2 fragmented cassette _C-terminal fragment were introduced into each of the strains of the iK4 gene fragmented in Example ⁇ 2-1> through homologous recombination to survive in the SC-URA minimal medium. Mutant strains in which the H P GAS2 gene was disrupted were obtained.
  • the present inventors compared the Hansenula polymorpha wild type DL1 strain with confocal microscopy and optical microscope to confirm the morphological characteristics of the HpGASl gene disruption strain and ⁇ 3 ⁇ 4 ⁇ 5 gene disruption strain prepared in ⁇ Example 2> .
  • the present inventors confirmed the physiological characteristics of the HpGASl gene disruption strain and ⁇ 5 gene disruption strain prepared in ⁇ Example 2> through the sensitivity to the cell wall inhibitor.
  • HpGASl gene disruption or H P GAS2 gene disruption Hansenula polymorpha DL1 wild type strain, HpGASl fragmented strain and HpGAS2 fragmented.
  • the strains were preincubated for 16 hours at 37 ° C at 200 rpm in 3 ml glucose 2% complex media each.
  • the cultured strains were first diluted with 1/10 of 1D in 1 ml of sterile water, and then serialized 1/10 for glucose 2% complex agar medium, 3% Calcofluor White (CFW), a cell wall synthesis inhibitor, 1% Congo Red (C) and osmotic perturbant 0.01% sodium dodecyl sulfate (SDS), respectively, were collected in a glucose 2% complex agar medium containing 1% and incubated at 37 ° C for 3 days.
  • C Calcofluor White
  • SDS osmotic perturbant 0.01% sodium dodecyl sulfate
  • HpGASl gene disruption strains were inhibited in 37% C and 42 0 C glucose 2% agar medium due to the change of cell wall structure, and wild type or even in cell wall synthesis inhibitor-sensitive media and osmotic disturbance-sensitive media. It was confirmed that growth was inhibited and growth rate was slow compared to H P GAS2 crushed strains (FIGS. 1, 2 and 3 of FIG. 9).
  • the inventors of the present invention have found that the cell wall following HpGASl gene disruption and HpGAS2 gene disruption is In order to confirm whether the total protein secretion is increased by the structural change was confirmed by the Bradford assay method.
  • Hanshenula polymorpha DL1 wild type strain,; 3 ⁇ 4 ⁇ A5 gene disruption strain and H P GAS2 gene disruption strain was pre-incubated for 16 hours at 37 ° C at 200 rpm in 3 ml glucose 2> complex medium 50 Incubated in ml glucose 2% complex medium to the initial 0D 600 value 0.3 to the same conditions. Take culture medium at each time of incubation
  • the present inventors confirmed whether the secretion of Glucose oxidase from the GOD producing strain was increased by HpGASl gene disruption.
  • wild type GOD producing strain H. poIymorpha / pIAMOX-GOO
  • HpGASl gene disruption strain polymorpha AffpGASl / pOLV X-GOm
  • HpGAS2 gene disruption strain HpGAS2 gene disruption strain
  • polymorpha o ⁇ 5 / pDLM0X-G0D (H) was incubated in 3 ml glucose 2% complex medium at 200 rpm for 16 hours at 37 ° C, and then the expression of the foreign recombinant protein expressed using the M0X promoter was determined.
  • Hazardous methane was inoculated in 50 ml of 2% complex medium to give an initial 0D 600 value of 0.3, followed by main culture.
  • 0D 600 corresponds to 2
  • the supernatant was secured by centrifugation at 4 ° C for 10 minutes at 13,000 rpm.
  • the supernatant was mixed with 5 ⁇ sample loading buffer, boiled for 5 minutes, and then electrophoresed in two pairs of 83 ⁇ 4> SDS-PAGE gels by dividing the sample in half (ie, the supernatant corresponding to 0D 600 value 1).
  • One of the two gels was stained with Coomassie Brilliant Blue R-250 Staining Solution (BIO-RAD) and bleached with destaining buffer.
  • the other gel was transferred to a PVDF membrane, and then the PVDF membrane was blocked with 5% skim milk solution, and the primary antibody His-probe (Santa Cruz Biotechnology) was diluted 1: 1000 and reacted for 2 hours. After reaction, wash three times for 10 minutes with TBST (50 mM Tris-HCKpH 7.5), 150 mM NaCl and 0.053 ⁇ 4> Tween 20), and then dilute the secondary antibody Anti-Mouse IgG (Sigma) to 1: 3000. The reaction was carried out for 30 minutes. Then, washed with TBST solution three times for 10 minutes and detected by ECL advanceTM Western Blotting Detect ion Kit (AmershamTM-GE Healthcare).
  • HSA secretion of recombinant human serum albumin (HSA) producing strain was increased by HpGASl and HpGAS2 gene disruption. It was. ' Specifically, wild type HSA producing strain (polyniorpha G3T8), HpGASl gene disruption strain (.polyniorpha G3T8 AHpGASl) and HpGAS2 gene disruption strain 07.
  • the supernatant was obtained by centrifugation at 4 ° C. for 10 minutes at 13,000 rpm.
  • the supernatant was mixed with 5 ⁇ sample loading buffer and boiled for less than 5 minutes, and then electrophoresed in two pairs of 1OT SDS—PAGfe gels by dividing the samples in half (ie, the supernatant corresponding to a 0D 600 value of 0.05).
  • One of the two gels was stained with Coomassie Brilliant Blue R_ 2 50 Staining Solution (BIO-RAD) and bleached with destaining buffer.
  • the other gel was transferred to a PVDF membrane, and the PVDF membrane was blocked with a 5% skim milk solution, and the first antibody, Anti-Human Albumin antibody (Sigma), was diluted 1: 10000 and reacted for 2 hours. After reaction, washed three times for 10 minutes with TBST (50 mM Tris-HCKpH 7.5), 150 mM NaCl and 0.05% Tween 20) solution, and then the second antibody, goat ant i -rabbit IgG-HRP (Santa Cruz Biotechnology) 1: Diluted to 5000 and reacted for 1 hour 30 minutes.
  • TBST 50 mM Tris-HCKpH 7.5
  • the present inventors have the same effect as the above experimental example by lacZ-URA3 ⁇ lacZ cassette
  • the lacZ ⁇ URA3-lacZ cassette was removed from the gene disruption strain.
  • chromosomal DNA isolated from Hanshenula polymorpha DL1 strain was used as a template to prepare lacZ-URA3-lacZ removal cassette using ⁇ AWJLForward (SEQ ID NO: 9) and ⁇ A _N—Reverse (SEQ ID NO: 10).
  • H P GAS1 ⁇ ⁇ C-terminal fragments (524 • bp) that could be fused with HpGASl ⁇ N-terminal fragments using A_C—Reverse (SEQ ID NO: 12) were obtained via polymerase chain reaction (PCR).
  • the present inventors produced vectors in which the HpGASl, HpGAS2 and ScGASl genes were cloned, respectively, and introduced them into the HpGASl gene disruption strain (.polyinorpha DL1 AHpGASl ⁇ ?).
  • HpGASl, HpGAS2 gene cassette and Saccharomyces cerevisiae amplified using primers of SEQ ID NOs: 17 and 18, and SEQ ID NOs: 19 and 20, respectively, as a template of chromosomal DNA isolated from Hanshenula polymorpha DL1 strain.
  • Chromosomal DNA isolated from BY4741 strain was amplified using primers of SEQ ID NOs: 21 and 22 as templates.
  • the ScGASl gene cassette was cloned into the blunt ends in the pDLGUK vector treated with restriction enzyme _ MssJ (FIG. 1).
  • the pDLGUK vector is a vector with genes ⁇ and ⁇ i? that can be randomly inserted near the telomeres of chromosomal DNA.
  • the transformant is capable of obtaining transformants that grow on SC-URA minimal media because the iffi gene is inserted. Number have.
  • HpGASrA inserted vector pDLGUK-y3 ⁇ 4 ⁇ 7A «
  • HpGAS27 ⁇ inserted vector MM-HpGAS2
  • ⁇ A inserted vector (pDLGUK- ScGASl) and pDLGUK blank were URA3 gene and H P GAS17 ⁇ crushed Hansenula polymorpha DL1 strain (.polymorpha DL1 ⁇ HpGAS ⁇ ⁇ 3) was transformed.
  • the present inventors confirmed the physiological characteristics due to the gene introduced in Experimental Example ⁇ 4-2> by integrating the culture medium diluted in a serial medium on a solid medium.
  • the strain supplemented with the HpGASl gene showed a similar degree of growth as the wild type (4 in FIG. 9), and ⁇ (strain supplemented with the 52 gene was slower than the wild type, but it was found to grow in a sensitive medium (FIG. 9).
  • Saccharomyces cerevisiae Strains supplemented with the GAS1 gene, ScGASl gene did not show growth growth, and showed the same growth as the strain into which the pDLGUK void was introduced (FIGS. 6 and 7 of FIG. 9).
  • scGASl-expressing vector or pDLGUK vaccinated strain was slightly increased than that of untransformed HpGASl crushed strain. It was judged as the same phenomenon reported to grow fast.
  • the present inventors also confirmed growth inhibition by cell wall attenuation by GAS1 gene disruption in Saccharomyces cerevisiae.
  • Saccharomyces cerevisiae strain C. cerevisiae BY4741 Saccharomyces cerevisiae strain C. cerevisiae BY4741
  • ScGASl crushed strain (5. cerevisiae BY4741 ⁇ 5 £) was pre-incubated in 3 ml glucose 2% complex medium at 200 rpm for 16 hours at 30 ° C.
  • the pre-incubated strains were serially maintained at OD 600 value 1 in 1 ml of sterile water. 1/10 dilutions, then 1% glucose 2% agar medium, 0.005% Congo Red (CR) and 0.01% sodium dodecyl sulfate (SDS) ⁇ Were accumulated and incubated for 3 days or 4 days at 30 ° C.
  • the present inventors produced vectors (YEp352ScGAPDHpt -HpGASl, YEp352ScGAPDHpt-HpGAS2), each of which was cloned into the above genes in order to confirm the function of the HpGASl and HpGAS2 genes using Saccharomyces cerevisiae strains. cerevisiae BY4741 AScGASD ⁇ ], respectively.
  • chromosomal DNA isolated from Hanshenula plymorpha DL1 strain Amplified using primers SEQ ID NO: 17 and 18, SEQ ID NO: 19 and 20, respectively, as a template
  • HpGASl and HpGAS2 gene cassettes were treated with restriction enzyme Xba / and cloned into YEp352ScGAPDHpt vector treated with restriction enzyme XZ / (FIG. 2).
  • the YEp352ScGAPDHpt vector is a vector with a 2 micron origin, a gene and a GAPDH promoter.
  • the transformant can be obtained from the SC-URA medium because the URA3 gene is inserted.
  • the HpGAS] 7 ⁇ inserted vector (YEp352ScGAPDHpt-HpGASl), the HpGAS27 ⁇ inserted vector (YEp352ScGAPDHpt-HpGAS2) and the YEp352ScGAPDHpt blank were scGASl crushed Saccharomyces cerevisiae strain (5. cerevisia SD BY474. Transformants were obtained from SC-URA minimal media, and diluted cell culture fluids were accumulated in sensitive solid media to compare growth.
  • Saccharomyces cerevisiae strain (5. cereP ' s / aeBY4741 ASc 5J / YEp352ScGAPI) Hp1, in which the 5rf 3/4 gene complementing the HpGASl gene was disrupted; -HpGASl) was recovered to a similar extent as wild type (5. cerevisiae BY4741) (Fig. 3, 3), and the strain complementing the HpGAS2 gene (5. cerevisiae BY4741 ASc / Y3 ⁇ 4) 352ScGAPDHpt—HpGAS2) was nocturnal (5. the growth was restored, so similar to the 's / aeBY4741) (4 in Fig.

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Abstract

The present invention relates to a method for using, in the production of exogenous recombinant protein, a variant microbial strain having a protein- secreting ability which is improved by damaging a protein of a novel kind. More specifically, the Hansenula polymorpha gene base sequence was screened so as to secure a group of candidates for an HpGAS gene coding for the Gas1 protein having a beta-1,3-glucanosyl transglycosylase action, and in this group a novel gene (HpGAS1) has been confirmed, and the gene in question was deleted so as to verify the function thereof, and as a result it has been confirmed that there are increased cell morphology changes and cell wall synthesis inhibitor susceptibility, increased total protein secretion, and markedly increased recombinant protein secretion, and thus the invention can advantageously be used as a resource for the production by secretion, the cleaning and the collection of exogenous recombinant protein.

Description

【명세서】  【Specification】
【발명의 명칭】  [Name of invention]
Hp GA S1 유전자 파쇄 효모 균주 및 이를 이용한 재조합 단백질의 생산 방법  Hp GA S1 Gene Fragmenting Yeast Strain and Method of Production of Recombinant Protein Using the Same
【기술분야】 i Technical Field i
본 발명은 신규 유전자를 파손시켜 단백질 분비능을 향상시킨 변이 균주 및 상기 변이 균주를 이용한 외래 재조합 단백질 생산 방법에 관한 것이다. 【배경기술】  The present invention relates to a mutant strain that breaks a novel gene to improve protein secretion ability and a foreign recombinant protein production method using the mutant strain. Background Art
단백질 의약품 개발은 1970년대에 유전자 재조합 기술이 보편화됨에 따라 시작되었다. 특히， 생산량이 한정된 호르몬， 항체 , 백신 및 생체기능성 단백질과 같은 유용 단백질을 미생물 숙주세포에서 대량 생산하여 의약품으로 개발하는 재조합 단백질 의약품 생산기술은 미생물을 세포공장 (Cell factory)으로 활용하는 대표적인 미생물 이용기술로 부상하고 있으며, 인류의 보건 복지가 향상됨에 따라 난치성 질환의 치료를 위한 고순도의 단백질성 의약품에 대한 수요가 기하급수적으로 증가되고 있어， 저렴한 비용으로 대량생산이 가능한 미생물 발현시스템을 이용한 재조합 의약품 생산기술 개발은 미래 의약산업의 성장에 크게 기여할 것으로 예측되고 있다. 초기 단백질 의약품은 인체 조직 또는 혈액에서 분리하는 경우가 있었으나, HIV와 간염바이러스를 비롯한 바이러스 감염 문제, 잠재적인 암 유발 인자의 잔류 가능성 등과 같은 심각한 문제들 때문에 현재  Protein drug development began in the 1970s with the introduction of genetic recombination technology. In particular, the recombinant protein pharmaceutical production technology, which produces a large amount of useful proteins such as hormones, antibodies, vaccines, and biofunctional proteins in a microbial host cell and develops them into pharmaceuticals, utilizes representative microorganisms that utilize microorganisms as cell factories. As the health and welfare of human beings improves, the demand for high-purity proteinaceous medicines for the treatment of intractable diseases is growing exponentially, and recombinant medicines using microbial expression systems capable of mass production at low cost Production technology development is expected to contribute greatly to the growth of the future pharmaceutical industry. Early protein drugs were sometimes isolated from human tissue or blood, but due to serious problems such as viral infections, including HIV and hepatitis, and the potential for residual cancer-causing agents,
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이들은 재 '조합 의약품으로 바뀌고 있으며， 고비용 기술인 동물세포에서 생산하는 경우에도 여전히 바이러스와 광우병의 원인이 되는 프라이온 등의 오염 가능성 문제가 남기 때문에 안전하다고 여겨지는 GRAS( generally recognized as safe) 미생물을 이용한 경제성 높은 재조합 단백질의 대량생산 기술개발의 중요성아 부각되고 있다. 미생물 활용 재조합 의약용 단백질 개발 분야의 경우 생산기술의 근간이 되는 신규 고발현 시스템 개발, 유전체 정보 기반의 고기능 숙주세포 재설계， 고성능 생산 균주의 대량 배양에 관한 분자생물공정 기술 개발 등아 국제싱장 개방과 함께 국가 경쟁력 강화를 위한 중요한 분야로 대두되고 있다. 재조합 단백질 생산 기술은 비교적 )근에 발달한 유전자 재조합 기술을 통하며 고등생물 유래의 유전자를 다양한 미생물로부터 대량 발현 생산하여 자연적으로 생산량이 한정된 고부가가치 의료용 단백질을 비교적 저렴한 비용으로 생산하는 기술로서 앞으로 난치성 질환의 증가와 국민 의료 수준의 제고에 따라 고 순도의 단백질성 의약품의 수요가 급증할 것으로 예상되고 있다. 따라서 인체에 무해한 다양한 미생물을 이용하여 신 기능성 재조합 단백질을 비교적 저렴한 비용으로 대량 생산할 수 있는 기술개발 연구가 필요한 실정이다. 효모를 숙주세포로 이용한 재조합 단백질생산 연구는 주로 전통효모 사카로마이세스 세레비지애 (5acc?a o/7y ?s cerevisiae)^ 이용하였으나 효율적인 외래단백질 발현을 위한 강력한 프로모터의 부재 또는 장시간 발효시에 야기되는 플라스미드의 불안정성 등의 이유로 재조합 단백질의 생산효율이 떨어지며， 고농도 배양시 fed-batch 발효 (ferment at ion)가 필요하고， 발현된 이종 단백질들이 하이퍼글라이코실레이션 (hyperglycosylation) 된다는 점에서 비적절한 숙주로 간주되고 있다 (Romanos, et al. , Yeast , 8， 423, 1992). 이와 같은 단점을 보완하는 외래단백질 발현 시스템이 메탄을 자화 효모인 피키아 파스토리스 ( / z/a / s/^r/s)에서 개발되었으며 (Sudbery et al. Yeast, 10， 1707, 1994； Cregg et al . , Bio/Technol . 5， 479， 1987)， 최근에는 또 다른.메탄을 자화 효모인 한세눌라 폴리모르파 0¾ 5«ί"/3 polyiwrpha)를 이용한 이종 단백질 발현 시스템의 개발 연구가 활발히 진행되고 있다 (Oh et al. , Biotechnol. J. 3, 1， 2008； Gellissen et al. , Bio/Technol . 9, 291, 1991； Janowicz et al . , Yeast 7， 431, 1991) . 새로운 대체숙주효모 중의 하나인 메탄올 자화 효모 한세눌라 폴리모르파 ( ¾ £//3 polymorphaV: 값싼 원료물질인 메탄올을 탄소원으로 이용하여 비교적 쉽게 고농도 배양이 가능하며， 메탄을 대사에 관련된 몇 가지 유전자 유래의 강력한 프로모터가 존재한다. 또한, 외래 유전자를 숙주세포의 염색체 DNA에 다중도입 (multicopy integration)할 수 있어 고농도 배양 중에도 :안정하게 유지되는 장점이 있다. 이에, 본 발명자들은 외래 재조합 단백질의 생산에 유리한 기존의 한세눌라 폴리모르파 (^ e/ /a polyworpha) 균주의 단백질 분비 생산을 더욱 증가시키기 위하여 세포벽 구성 및 투과성 결정에 증요한 신규한 베타 -1 , 3-글루카노실트랜스글라이코실레이즈 (beta-1 ,3-glucanosyl transglycosy lase ) 활성을 가지는 HpGASl 유전자를 확보하였으며， 상기 유전자 결손시 외래 재조합 단백질 분비능이 증가함을 확인하였으므로， 이를 통해 본 발명의 HpGASl 유전자가 단일 결손된 한세눌라 폴리모르파 변이 균주가 의약용 재조합 단백질의 분비 생산을 위한 자원으로서 유용하게 이용될 수 있음을 밝힘으로써, 본 발명을 완성하였다. They are turning into ' recombinant drugs ' , and even if they are produced in animal cells, which are expensive technologies, they still use GRAS (generally recognized as safe) microorganisms, which are considered safe because of the potential for contamination such as prion, which causes viruses and mad cow disease. The importance of development of mass production technology of economical recombinant protein has been highlighted. In the field of recombinant pharmaceutical protein development using microorganism, development of new high expression system which is the basis of production technology, redesign of high function host cell based on genomic information, The development of molecular bioprocessing technology for mass cultivation of high-performance producing strains is emerging as an important field for strengthening national competitiveness. Recombinant protein production technology is a technology that produces high value-added medical proteins with limited production cost by producing genes derived from higher organisms from a variety of microorganisms through gene recombination technology developed relatively). The demand for high-purity proteinaceous drugs is expected to soar due to the increase of diseases and the improvement of national medical standards. Therefore, there is a need for a technology development research capable of mass production of new functional recombinant protein at a relatively low cost using various microorganisms that are harmless to the human body. Recombinant protein production studies using yeast as a host cell mainly used the traditional yeast Saccharomyces cerevisiae, but the absence of a strong promoter for efficient foreign protein expression or long-term fermentation Due to the instability of the plasmid, the production efficiency of recombinant protein is low, fed-batch fermentation is required in high concentration culture, and the heterologous proteins expressed are hyperglycosylated. (Romanos, et al., Yeast, 8, 423, 1992). A foreign protein expression system that compensates for these drawbacks was developed in Pichia pastoris (/ z / a / s / ^ r / s), a magnetizing yeast methane (Sudbery et al. Yeast, 10, 1707, 1994; Cregg et al., Bio / Technol. 5, 479, 1987), and recently, the development of heterologous protein expression systems using Hansenula polymorpha 0¾ 5 «ί” / 3 polyiwrpha, a magnetizing yeast of methane, has been actively studied. (Oh et al., Biotechnol. J. 3, 1, 2008; Gellissen et al., Bio / Technol. 9, 291, 1991; Janowicz et al., Yeast 7, 431, 1991). One of the yeasts, methanol magnetization yeast Hansenula polymorphaV (¾ £ / / 3 polymorphaV): It is possible to cultivate relatively high concentrations easily using methanol, a cheap raw material as a carbon source, and a strong promoter derived from several genes involved in metabolism. In addition, the foreign gene is transferred to the host cell's chromosomal DNA. Multicopy It can be integrated) even during high concentration cultivation : it has the advantage of keeping stable. Accordingly, the present inventors have proposed a novel beta-1 that is critical for cell wall composition and permeability determination in order to further increase the production of protein secretion of the existing Hansenula polymorpho (^ e / / a polyworpha) strain, which is advantageous for the production of foreign recombinant proteins. , HpGASl gene having 3-glucanosyl transglycosy lase (beta-1, 3-glucanosyl transglycosy lase) activity was secured, it was confirmed that the foreign recombinant protein secretion ability increases when the gene is deleted, through the present invention The present invention was completed by revealing that a single-deleted Hansenula polymorpha mutant strain having a single deletion of HpGASl gene can be usefully used as a resource for secretory production of a pharmaceutical recombinant protein.
【발명의 상세한 설명】 [Detailed Description of the Invention]
[기술적 과제]  [Technical Challenges]
본 발명의 목적은 서열번호 1로 기재되는 HpGASl 유전자가 결손된 단백질 분비능이 향상된 기탁번호 KCTC12220BP의 한세눌라 폴리모르파 (Hansenula polymorpha) 변이 균주를 제공하는 것이다. ；  It is an object of the present invention to provide a Hansenula polymorpha variant strain of Accession No. KCTC12220BP with improved protein secretion ability in which the HpGASl gene described in SEQ ID NO: 1 is deleted. ;
또한， 본 발명의 또 다른 목적은 단백질 분비능이 향상된 효모 균주의 제조 방법을 제공하는 것이다.  In addition, another object of the present invention to provide a method for producing a yeast strain with improved protein secretion ability.
또한， 본 발명의 또 다른 목적은 외래 재조합 단백질 생산 방법을 제공하는 것이다.  In addition, another object of the present invention is to provide a foreign recombinant protein production method.
아을러， 본 발명의 또 다른 목적은 단백질 분비능을 향상시키기 위한, 서열번호 1로 기재되는 HpGASl 유전자가 결손된 한세눌라 폴리모르파 Ofe^ey / polymorpha) 변이 균주의 용도를 제공하는 것이다.  In addition, another object of the present invention is to provide a use of a Hanshenula polymorpho Ofe ^ ey / polymorpha) strain that is deficient in the HpGASl gene as set forth in SEQ ID NO: 1 to enhance protein secretion ability.
【기술적 해결방법】 Technical Solution
상기 목적을 달성하기 위하여， 본 발명은 서열번호 1로 기재되는 HpGASl 유전자가 결손된 단백질 분비능이 향상된 기탁번호 KCTC12220BP의 한세눌라 폴리모르파 0¾ 7se //a polyworpha) 변이 균주를 제공한다. In order to achieve the above object, the present invention is HpGASl described in SEQ ID NO: 1 It provides a Hanshenula polymorpha 0¾ 7se // a polyworpha) variant strain of Accession No. KCTC12220BP with improved gene secretion ability.
또한， 본 발명은 단백질 분비능이 향상된 효모 균주의 제조 방법을 제공한다.  The present invention also provides a method for producing a yeast strain with improved protein secretion ability.
또한， 본 발명은 외래 재조합 단백질 생산 방법을 제공한다.  The present invention also provides a foreign recombinant protein production method.
아울러， 본 발명은 단백질 분비능을 향상시키기 위한,서열번호 1로 기재되는 HpGASl 유전자가 결손된 한세눌라 폴리모르파 0¾72s ¾//a poly orpha) 변이 균주의 용도를 제공한다. 【유리한 효과】  In addition, the present invention provides a use of a Hansennura polymorpha 0¾72s ¾ // a poly orpha) mutant strain lacking the HpGASl gene as set forth in SEQ ID NO: 1 for improving protein secretion ability. Advantageous Effects
본 발명의 한세눌라 폴리모르파 0¾/2se //a polymorpha)^ 베타— 1,3-글루카노실트랜스글라이코실레이즈 (beta_l,3-glucanosyltransglycosylase )를 코딩하는 신규한 유전자 HpGASl^ 단일 결손시킨 한세눌라 폴리모르파 균주는 세포의 형태가 변화했으며， 세포벽 합성 저해제에 대한 감수성이 증대되었고， 총 단백질 분비량이 증대됨을 확인하였으며， 특히, 재조합 단백질의 분비량 및 활성이 현격히 향상됨을 확인하였으므로， 외래 재조합 단백질의 분비생산， 정제 및 수확에 유리하여 재조합 단백질의 생산을 위한 자원으로서 유용하게 이용될 수 있다.  Hansenula polymorpha 0¾ / 2se // a polymorpha) ^ beta—the novel gene HpGASl ^ single-deleted gene encoding the 1,3-glucanosyltransglycosylase (beta_l, 3-glucanosyltransglycosylase) of the present invention As the Nula polymorpha strains changed cell morphology, increased sensitivity to cell wall synthesis inhibitors, increased total protein secretion, and in particular, significantly increased secretion and activity of recombinant protein. It can be usefully used as a resource for the production of recombinant protein in the production, purification and harvesting of.
【도면의 간단한 설명】 [Brief Description of Drawings]
도 1은 서열번호 1로 기재되는 HpGASl 유전자의 서열을 NCBI의 BLAST를 이용하여 상동성을 검색한 결과이다.  1 is a result of searching for homology to the sequence of the HpGASl gene described in SEQ ID NO: 1 using BLAST of NCBI.
도 2는 서열번호 2로 기재되는 HpGAS2 유전자의 서열을 NCBI의 BLAST를 이용하여 상동성을 검색한 결과이다.  Figure 2 shows the results of searching for homology to the sequence of the HpGAS2 gene described in SEQ ID NO: 2 using BLAST of NCBI.
도 3은 ScGasl 도메인을 통해 HpGasl 도메인을 비교 분석한 도이다:  3 is a comparative analysis of the HpGasl domain via the ScGasl domain:
*: Potential N-glycosylat ion 위치;  *: Potential N-glycosylat ion position;
ᅀ: GPI 부착 위치 (GPI attachment sites);  VIII: GPI attachment sites;
▲: Strict ly conserved residues in the catalytic domain; 1-22： Signal sequence； ▲: Strict ly conserved residues in the catalytic domain; 1-22: Signal sequence;
23—332: -(l,3)-glucan transferase domain (GluTD)；  23—332:-(l, 3) -glucan transferase domain (GluTD);
370-462： 8 Cys-region;  370-462: 8 Cys-region;
485-525： Ser-r ich region; 및  485-525: Ser-r ich region; And
537-559： 소수성 부위 (Hydrophobic region).  537-559: Hydrophobic region (Hydrophobic region).
도 4는 腦 유전자 파쇄 카세트의 제작 과정을 나타내는 도이다.  Figure 4 is a diagram showing the manufacturing process of the 腦 gene disruption cassette.
도 5는 HpGASl유전자 파쇄 카세트의 제작 과정을 나타내는 도이다.  5 is a diagram illustrating a manufacturing process of the HpGASl gene disruption cassette.
도 6은 ^ 5 유전자 파쇄 카세트의 제작 과정을 나타내는 도이다.  6 is a diagram showing the manufacturing process of the ^ 5 gene disruption cassette.
도 7은 HpGASl, HpGAS2및 ScGASl유전자를 클로닝한 한세눌라 폴리모르파용 백터와사카로마이세스 세레비지애용 백터이다:  7 is a vector for Hanshenula polymorpha and a Saccharomyces cerevisiae vector cloned with HpGASl, HpGAS2 and ScGASl genes:
1: 한세눌라 폴리모르파용 백터 (pDLGUK):  1: Vector for Hansenula polymorpha (pDLGUK):
2： .사카로마이세스 세레비지애용 백터 (YEp352ScGAPDHpt ) .  2: Vector for Saccharomyces cerevisiae (YEp352ScGAPDHpt).
도 8은 HpGASl 및 HpGAS2단일 결손 변이주의 세포 형태적 특징올 보여주는 사진이다:  FIG. 8 is a photograph showing the cell morphological features of HpGASl and HpGAS2 single deletion mutants.
A: 2% 포도당 최소 배지에서 48 시간 동안 배양한 후 2000 배율의 공초점현미경으로 확인한사진;  A: Photographed by confocal microscopy at 2000 magnification after incubation for 48 hours in 2% glucose minimal medium;
B: 2% 포도당 복합 배지에서 48 시간 동안 배양한 후 2000 배율의 공초점현미경으로 확인한사진;  B: Photograph confirmed by confocal microscope at 2000 magnification after incubation for 48 hours in 2% glucose complex medium;
C: 2% 포도당 복합 배지에서 48 시간 동안 배양한 후 1000 배율의 광학현미경으로 확인한 사진;  C: Photographed with an optical microscope at 1000 magnification after incubation for 48 hours in 2% glucose complex medium;
1: 한세눌라 폴리모르파 야생형 균주 07. polymorpha DL1)；  1: Hansenula polymorpha wild type strain 07. polymorpha DL1);
2: HpGASl 유전자가 결손된 한세눌라 폴리모르파 균주 ( . polymorpha DL1 AHpGASl)； 및  2: Hanshenula polymorpha strain lacking HpGASl gene (.polymorpha DL1 AHpGASl); and
3： HpGAS2 유전자가 결손된 한세눌라 폴리모르파 균주 0¥. polymorpha DL1 AHpGAS2) . 도 9는 HpGASl 및 HPGAS2 단일 결손 변이 균주， 및 유전자 회복 균주의 생리적 특징을 확인한사진이다: 3: Hansenula polymorpha strain with a lack of HpGAS2 gene 0 ¥. polymorpha DL1 AHpGAS2). 9 shows HpGASl and H P GAS2 single deletion variant strain, and gene recovery strain Here are some pictures of physiological features:
A: 2%포도당 복합 배지 (370C); B: 2% 포도당 복합 배지 (42°C); A: 2% glucose complex medium (37 0 C); B: 2% glucose complex medium (42 ° C.);
C: Sodium Dodecyl Sulfate (SDS) - 2%포도당 복합 배지 ; C: Sodium Dodecyl Sulfate (SDS)-2% Glucose Complex Medium;
D: Congo Red (CR) - 2%포도당 복합 배지 ;  D: Congo Red (CR) -2% Glucose Complex Medium;
E: Calcofluor White (CFW) - 2%포도당 복합 배지;  E: Calcofluor White (CFW) -2% glucose complex medium;
1： 한세눌라 폴리모 Ξ파 DL1 야생형 균주 ( . polymorpha DLl)；  1: Hanshenula polymo Ξpa DL1 wild type strain (.polymorpha DLl);
2： HpGASl 유전자가 결손된 한세눌라 폴리모르파 균주 ( . polymorpha DLl AHpGASl) 3： HpGAS2 유전자가 결손된 한세눌라 폴리모르파 균주 ( polymorpha DLl 2: Hanshenula polymorpha strain lacking HpGASl gene (.polymorpha DLl AHpGASl) 3: Hanshenula polymorpha strain lacking HpGAS2 gene (polymorpha DLl)
AHpGAS2)； AHpGAS2);
4: HpGASl 발현백터 (pDLGUK-HpGASl)로 형질전환된 HpGASl 유전자가 결손된 한세눌라 폴리모르파 균주 ( polymorpha DLl AHpGASl ^¾¾5/pDLGUK-HpGASl)； 4: Hanshenula polymorpha strain (polymorpha DLl AHpGASl ^ ¾¾5 / pDLGUK-HpGASl) lacking the HpGASl gene transformed with HpGASl expression vector (pDLGUK-HpGASl);
5： HpGAS2 발현백터 (PDLGUK-HpGAS2)로 형질전환된 HpGASl 유전자가 결손된 한세눌라 폴리모르파 균주 ( . polymorpha DLl AHpGASl ^^j pDLGUK-HpGAS2)； 5: Hansenula polymorpha strain lacking the HpGASl gene transformed with HpGAS2 expression vector ( P DLGUK-HpGAS2) (.polymorpha DLl AHpGASl ^^ j pDLGUK-HpGAS2);
6: ScGASl 발현백터 (pDLGUK-ScGASl)로 형질전환된 HpGASl 유전자가 결손된 한세눌라 폴리모르파 균주 ( polymorpha DLl AHpGASl Zl« 5/pDLGUKᅳ ScGASl )； 및6: Hansenula polymorpha strain (polymorpha DLl AHpGASl Zl «5 / pDLGUK ᅳ ScGASl) that lacks the HpGASl gene transformed with the ScGASl expression vector (pDLGUK-ScGASl); and
7: 대조군인 공백터 (pDLGUK)로 형질전환된 HpGASl유전자가 결손된 한세눌라 폴리모르파 균주 ( polymorpha DLl AHpGASl AURAS/ pOLQ\ ) . 도 10은 HpGASl 및 HPGAS2단일 결손 변이 균주에서의 총 단백질 분비량을 비교한 그래프이다: 7: Hanshenula polymorpha strain (polymorpha DLl AHpGASl AURAS / pOLQ \) lacking the HpGASl gene transformed with the control site pDLGUK. 10 is a graph comparing total protein secretion in HpGASl and H P GAS2 single deletion variant strains:
A: 한세눌라 폴리모르파 DL1 야생형 균주 ( . polymorpha DLl);  A: Hansenula polymorpha DL1 wild type strain (.polymorpha DLl);
B: HpGASl올 결손시킨 DLl 균주 0. polymorpha DLl AHpGASl)； 및  B: DLl strain deficient in HpGASl 0. polymorpha DLl AHpGASl); and
C: HpGAS 결손시킨 DLl 균주 ( . polymorpha DLl AHPGAS2) . 도 11은 HpGASl 및 HpGAS2단일 결손 변이 균주에서 Glucose oxidase (GOD) 분비량을 비교한사진이다: C: HpGAS deficient DLl strain (.polymorpha DLl AH P GAS2). FIG. 11 is a photograph comparing Glucose oxidase (GOD) secretion in HpGASl and HpGAS2 single deletion strains:
M: 단백질 크기 표지  M: protein size label
1： 야생형 GOD 생산균주 [ poJy orpAa/^lMOX-GOOi )]；  1: wild type GOD producing strain [poJy orpAa / ^ lMOX-GOOi]];
2： HpGASl^ 결손시킨 GOD 생산균주 [ polymorpha ^i¾5i/pDLM0X-G0D(H)]；  2: HpGASl ^ deficient GOD producing strain [polymorpha ^ i¾5i / pDLM0X-G0D (H)];
3： ffpGAS 결손시킨 GOD 생산균주 [ . polymorpha Λ¾？ AS /pDLMOX-GOD(H)] . 도 12는 HpGASl 및 ^O 1 단일 결손 변이주에서 분비된 Glucose oxidase3: GOD-producing strains lacking ffpGAS [. polymorpha Λ¾？ AS / pDLMOX-GOD (H)]. Figure 12 Glucose oxidase secreted from HpGASl and ^ O 1 single deletion mutants
(GOD) 활성을 비교한 그래프이다: (GOD) is a graph comparing activity:
1： 야생형 GOD 생산균주 [H. poIymorp/ia/pOL OX~GOO(E)]; 1 ： Wild type GOD producing strain [H. poIymorp / ia / pOL OX - GOO (E)];
2： HpGASl^ 결손시킨 GOD 생산균주 [ polymorpha ^i¾52/pDLM0X-G0D(H)]；  2: HpGASl ^ deficient GOD producing strain [polymorpha ^ i¾52 / pDLM0X-G0D (H)];
3： HpGAS 결손시킨 GOD 생산균주 [ polymorpha AffpGAS pOLUOI-GODiR)] . 도 13은 HpGASl 및 HpGAS2 단일 결손 변이 균주에서 Human serum albumin (HSA) 분비량을 비교한사진이다: 3: HpGAS-deficient GOD producing strain [polymorpha AffpGAS pOLUOI-GODiR)]. Figure 13 is a photograph comparing human serum albumin (HSA) secretion in HpGASl and HpGAS2 single deletion strains:
M: 단백질 크기 표지  M: protein size label
1: 야생형 HSA 생산균주 ( . polymorpha G3T8);  1: wild type HSA producing strain (.polymorpha G3T8);
2： HpGASl^ 결손시킨 HSA 생산균주 ( . polymorpha G3T8 AHpGASl)； 및  2: HSA producing strain HpGASl ^ deficient (.polymorpha G3T8 AHpGASl); and
3： HpGAS 결손시킨 HSA 생산균주 ( . polymorpha G3T8 AHpGAS2) . 도 14는 HpGASl유전자 파쇄된 균주와 HPUR/ 결손시키는 과정을 나타내는 도이다. ' 3: HSA producing strains deficient in HpGAS (.polymorpha G3T8 AHpGAS2). 14 is a diagram showing a process of H PGASl gene disrupted strains and H P UR / deletion. '
도 15는 ScGASl 유전자가 결손된 사카로마이세스 세레비지애균주 (5. cerevisiae BY4741 에서 HpGASl 및 ¾?fi45 유전자 발현백터를 형질전환한 균주의 생리적 특징을 확인한 사진이다:  Figure 15 is a photograph confirming the physiological characteristics of strains transformed HpGASl and ¾? Fi45 gene expression vector in 5. cerevisiae BY4741 lacking the ScGASl gene:
A: 2%포도당 복합배지； B: Sodium Dodecyl Sulfate (SDS)ᅳ 2%갈락토오즈 및 1%라피노오즈 복합 배지； C: Congo Red (CR) - 2% 갈락토오즈 및 1% 라피노오즈 최소 배지 ; A: 2% glucose complex medium; B: Sodium Dodecyl Sulfate (SDS) ᅳ 2% galactose and 1% raffinose complex medium; C: Congo Red (CR)-2% galactose and 1% raffinose minimal medium;
1: 사카로마이세스 세레비지애 야생형균주 (5. cerevisiae BY4741);  1: Saccharomyces cerevisiae wild type strain (5. cerevisiae BY4741);
2： ScGASl 유전자가 결손된 사카로마이세스 세레비지애 균주 ( cerevisiae BY4741 AScGASl)；  2: Saccharomyces cerevisiae strain lacking ScGASl gene (cerevisiae BY4741 AScGASl);
3: HpGASl 발현 백터 (YEp352ScGAPDHpt-HpGASl) 로 형질전환된 ScGASl 유전자가 결손된 사카로마이세스 세레비지애 균주 (5. cerevisiae BY4741 15^5i/YEP352ScGAPDHpt -HpGASl)； 3: Saccharomyces cerevisiae strain lacking ScGASl gene transformed with HpGASl expression vector (YEp352ScGAPDHpt-HpGASl) (5. cerevisiae BY4741 15 ^ 5i / YE P 352ScGAPDHpt -HpGASl);
4： ^ A 발현백터 (YEp352ScGAPDHpt-HpGAS2)로 형질전환된 5 45？유전자가 결손된 사카로마이세스 세레비지애 균주 (5. cerevisiae BY4741 15c^5i/YEp352ScGAPDHpt -HpGAS2 )； 및  4: Saccharomyces cerevisiae strain (5. cerevisiae BY4741 15c ^ 5i / YEp352ScGAPDHpt -HpGAS2) that lacks the 5 45? Gene transformed with ^ A expression vector (YEp352ScGAPDHpt-HpGAS2); and
5： 대조군인 공백터 (YEp352ScGAPDHpt)로 형질전환된 ScGASl유전자가 결손된 사카로마이세스 세레비지애 균주 ( S. cerevisiae BY4741 l&i¾5i/YEp352ScGAPDHpt ) . 5: Saccharomyces cerevisiae strain (S. cerevisiae BY4741 l & i¾5i / YEp352ScGAPDHpt), which lacks the ScGASl gene transformed with the control group, empty site (YEp352ScGAPDHpt).
【발명의 실시를 위한 형태】 [Form for implementation of invention]
이하， 본 발명을 상세히 설명한다. 본 발명은 서열번호 1로 기재되는 HpGASl 유전자가 결손된 단백질 분비능이 향상된 가탁번호 KCTC12220BP의 한세눌라 폴리모르파 (^;?se //a polymorpha) 변이 균주를 제공한다. 본 발명의 구체적인 실시예에서， 본 발명자들은 한세눌라 폴리모르파 DL1 유전체 정보 데이터베이스에서 다른 효모의 Gasl 단백질과 아미노산 서열이 유사한 단백질을 번역하는 HpGAS 유전자 후보들을 확보하였다. 그 결과 2 종의 한세눌라 폴리모파 유전자들 (^ί 5Λ HpGAS2)o] 타 효모의 Gasl 단백질과 아미노산 서열 상동성이 높은 단백질을 번역하는 것으로 분석되었다 (도 1과 2 참조). 그 중 사카로마이세스 세레비지애의 ScGasl 단백질의 아미노산 서열과 더 높은 상동성을 갖는 단백질을 번역하는 신규한 서열의 HpGASl유전자 번역하는 단백질의 아미노산 서열에 여러 Gasl 단백질에 보존적인 아미노산 서열 및 활성 도메인이 존재함을 확인하였다 (도 3참조). HpGASl^ ^ί 5 유전자가 파쇄된 균주를 선별하기 위하여 우선， 파쇄 카세트로 한세눌라 폴리모르파의 U A3 유전자를 파쇄한 뒤， HpGASl 및 HPGAS2유전자 파쇄 카세트를 이용한 상동재조합을 통해 한세눌라 폴리모르파 균주의 HpGASl 및 HPGAS2유전자를 각각 파쇄하였다. HpGASl 및 HpGAS2유전자가 파쇄된 한세눌라 폴리모르파 돌연변이 균주들의 특성을 비교 분석한 결과 HpGASl 파쇄 균주만이 GAS1 유전자 파쇄 균주의 특성을 보였다. HpGASl 유전자가 파쇄된 한세눌라 폴리모르파 균주에서 총 단백질 분비량이 증가함을 확인하였으며 (도 10 참조), 외래 재조합 단백질인 glucose oxidase (GOD) (Kim et al . , Glycobiology . 14, 243, 2004)및 human serum albumin (HSA) (Kang et al . , Biotechnol Bioeng. 76， 175, 2001)를 분비생산하는 균주의 HpGASl 유전자를 파쇄한 결과 해당 재조합 단백질의 분비량이 현격하게 증가함을 확인할 수 있었다 (도 11， 도 12 및 도 13 참조). 아울러， 상기 HpGASl 유전자 발현 백터를 형질전환하여 HpGASl 결손 돌연변이를 회복시킨 경우, 유전자 파쇄시 관찰된 감수성 배지에서의 생장 저하가 복원되는 것을 알수 있었다 (도 9 및 도 15 참조). 따라서， HpGASl유전자를 파쇄한 한세눌라 폴리모르파 균주의 단백질 분비능의 향상은; HpGASl 유전자의 단일 결실에 의한 것임을 재차 확인할 수 있었다. 본 발명은 Hereinafter, the present invention will be described in detail. The present invention provides Hansenula polymorpha (^ ;? se // a polymorpha) mutant strain of Accession No. KCTC12220BP with improved protein secretion ability, wherein the HpGASl gene described in SEQ ID NO: 1 is deleted. In a specific embodiment of the present invention, the inventors obtained HpGAS gene candidates for translating proteins having amino acid sequences similar to those of other yeast Gasl proteins in the Hanshenula polymorpha DL1 genomic information database. As a result, two Hansenula polymorpha genes (^ ί 5Λ HpGAS2) o] were analyzed to translate Gasl proteins of other yeasts and proteins having high amino acid sequence homology (see FIGS. 1 and 2). among them HpGASl gene of a novel sequence that translates a protein having higher homology with the amino acid sequence of ScGasl protein of Saccharomyces cerevisiae The amino acid sequence and the active domain conserved in several Gasl proteins exist in the amino acid sequence of the translating protein It was confirmed that (see Figure 3). In order to screen for strains in which the HpGASl ^ ^ ί 5 gene was disrupted, first, the Hansenula polymorpha U A3 gene was disrupted with a disruption cassette, followed by homologous recombination using HpGASl and H P GAS2 gene disruption cassettes. The HpGASl and H P GAS2 genes of the morpha strains were respectively disrupted. As a result of comparing and analyzing the characteristics of Hansenula polymorpha mutant strains in which the HpGASl and HpGAS2 genes were crushed, only the HpGASl crushed strain showed the characteristics of the GAS1 gene crushed strain. It was confirmed that the total protein secretion was increased in the Hansenula polymorpho strains in which the HpGASl gene was disrupted (see FIG. 10), and the foreign recombinant protein glucose oxidase (GOD) (Kim et al., Glycobiology. 14, 243, 2004). And disruption of the HpGASl gene of strains that secrete and produce human serum albumin (HSA) (Kang et al., Biotechnol Bioeng. 76, 175, 2001). 11, see FIG. 12 and FIG. 13). In addition, when the HpGASl gene expression vector was transformed to recover the HpGASl deletion mutant, it was found that the growth decrease in the sensitive medium observed during gene disruption was restored (see FIGS. 9 and 15). Therefore, the improvement of the protein secretion ability of Hanshenula polymorpho strain which crushed the HpGASl gene ; It was confirmed again that it was due to a single deletion of the HpGASl gene. The present invention
1) URA3 유전자 파쇄 카세트를 상동재조합을 통해 효모 균주에 도입하는 단계; 및  1) introducing the URA3 gene disruption cassette into the yeast strain through homologous recombination; And
2) 상기 균주에 HpGASl 유전자 파쇄 카세트를 상동재조합을 통해 도입하는 단계를 포함하는 단백질 분비능이 향상된 효모 균주의 제조 방법을 제공한다.  2) provides a method for producing a yeast strain with improved protein secretion capacity comprising the step of introducing the HpGASl gene disruption cassette to the strain through homologous recombination.
상기 画 유전자 파쇄 카세트는 HpURA3 유전자의 N 말단 절편， HpURA3 유전자의 C 말단 절편 및 Zeocin 저항성 유전자 (ZeoR) 카세트를 이용하여 제작한 파쇄카세트인 것이 바람직하나 이에 한정되지 않는다. The 画 gene disruption cassette was prepared using an N-terminal fragment of the HpURA3 gene, a C-terminal fragment of the HpURA3 gene, and a Zeocin resistance gene (Zeo R ) cassette. It is preferably a crushed cassette, but is not limited thereto.
상기 HpGASl 유전자 파쇄 카세트는 서열번호 1로 기재되는 염기서열로 구성되는 HpGASl 유전자의 N 말단 절편， HpGASl 유전자의 C 말단 절편 및 7ac -y¾? 카세트의 N-말단 절편， 1 acZᅳ HpURA3- 1 acZ？씨트 C-말단 절편 를 이용하여 제작한 파쇄 카세트인 것이 바람직하나 이에 한정되지 않는다. 본 발명의 구체적인 실시예에서, 본 발명자들은 유전자 파쇄를 구별할 수 있도록 腿3 유전자를 파쇄하기 위하여， HPURA3 유전자의 N 말단 절편, HpUM3 유전자의 C말단 절편 및 Zeocin저항성 유전자 (ZeoR) 카세트를 이용하여 « 파쇄 카세트를 제작하여 한세눌라 폴리모르파 균주에 형질전환한 후 상동재조합 방법으로 염색체상의 해당 유전자를 파쇄하였다 (도 4 참조). 유전자가 파쇄된 균주에 HpGASl 유전자의 N 말단 절편， HpGASl 유전자의 C 말단 절편 및 lacZ-HpUM3-lacZ 7\꼐트^ N-말단 절편 , 1 acZ-HpURA3- 1 acZ ?\ Ο말단 절편을 이용하여 제작한 파쇄카세트를 형질전환하여 HpGASl 유전자가 파쇄된 균주를 제작하였으며 (도 5참조)， ^i S 유전자가 파쇄된 균주의 단백질 분비능이 향상됨을 확인하였다. 이를 통해， 기존의 한세눌라 폴리모르파 야생형 균주 및 특정 재조합 단백질을 분비하는 야생형의 재조합 한세눌라 폴리모르파 균주의 HpGASl 유전자를 파쇄함으로써 단백질 분비가 월등히 증가한 한세눌라 폴리모르파 균주를 제작할 수 있었다. 본 발명은 The HpGASl gene disruption cassette comprises an N-terminal fragment of the HpGASl gene, a C-terminal fragment of the HpGASl gene, and 7ac -y¾? N-terminal fragment of cassette, 1 acZ ᅳ HpURA3- 1 acZ? It is preferable to use a shredding cassette produced using a sheet C-terminal fragment, but is not limited thereto. In a specific embodiment of the present invention, the inventors of the present invention, in order to disrupt the 腿 3 gene to distinguish gene disruption, the N terminal segment of the H P URA3 gene, the C terminal segment of the HpUM3 gene and Zeocin resistance gene (Zeo R ) cassette Using the «fragment cassette was transformed to Hanshenula polymorpha strain and then the corresponding gene on the chromosome was disrupted by homologous recombination method (see Fig. 4). The N-terminal fragment of the HpGASl gene, the C-terminal fragment of the HpGASl gene, lacZ-HpUM3-lacZ 7 \ hent ^ N-terminus fragment, and 1 acZ-HpURA3- 1 acZ? \ Ο terminal fragment were prepared in the fragmented strain. One disrupted cassette was transformed to prepare a strain in which the HpGASl gene was crushed (see FIG. 5), and it was confirmed that the protein secretion ability of the strain in which the ^ i S gene was crushed was improved. Through this, by breaking up the HpGASl gene of the conventional Hanshenula polymorpha wild type strain and wild type recombinant Hanshenula polymorpha strain secreting a specific recombinant protein, it was possible to produce a Hanshenula polymorpha strain with a significantly increased protein secretion. The present invention
1) HpGASl 유전자가 파쇄된 상기 변이 균주에 목적 단백질을 코딩하는 유전자를 포함하는 백터를 도입시켜 재조합 효모 균주를 제조하는 단계; 및  1) preparing a recombinant yeast strain by introducing a vector comprising a gene encoding a protein of interest into the mutant strain from which the HpGASl gene has been disrupted; And
2) 상기 재조합 효모 균주를 배양하는 단계를 포함하는 외래 재조합 단백질 생산 방법을 제공한다.  2) provides a foreign recombinant protein production method comprising the step of culturing the recombinant yeast strain.
상기 재조합 효모 균주는 사카로마이세스 (5ac ¾/ ¾z ^es), 클루베로마이入入스 Uduyveroinyces ， 피키 \iPichia)， 한세눌라 0¾/7se // ) 또는 캔디다 속으로 이루어진 군으로부터 선택되는 어느 하나인 것이 바람직하며， 한세눌라 폴리모르파 균주인 것이 더욱 바람직하나 이에 한정되지 않는다. The recombinant yeast strain is Saccharomyces (5ac ¾ / ¾z ^ es), Kluberomyces Uduyveroinyces, Picky \ iPichia, Hanshenula 0¾ / 7se //) or It is preferably any one selected from the group consisting of Candida genus, and more preferably, but not limited to Hanshenula polymorpha strain.
상기 외래 재조합 생산 방법은 생물반웅기 (bioreactor)를 이용하여 상기 변이주를 대량 배양하는 단계를 추가로 포함하는 것이 바람직하나 이에 한정되지 않는다. 본 발명의 구체적인 실시예에서， 본 발명자들은 glucose oxidase(GOD) 또는 인간 혈청 알부민 (HSA)을 코딩하는 유전자를 포함하는 백터가 도입되어 외래 재조합 단백질을 생산하는 GOD 생산균주 및 HSA 생산균주의 HpGASl 또는 HpGAS2 유전자를 파쇄시킨 뒤 배양한 결과， 특히 HpGASl 유전자 파쇄의 경우 상기 외래 재조합 단백질의 분비능이 월등히 증가하였으며， 생산된 단백질의 활성 또한 증가함을 확인하였다 (도 11， 도 12 및 도 13). 따라서， 본 발명의 HpGASl유전자를 단일 파쇄시킨 한세눌라 폴리모르파 균주를 외래 재조합 단백질의 생산을 위한 자원으로서 유용하게 이용될 수 있다. 아울러,본 발명은 단백질 분비능을 향상시키기 위한,서열번호 1로 기재되는 HpGASl 유전자가 결손된 한세눌라 폴리모르파 ( ? /7«? /3 polymorpha) 변이 균주의 용도를 제공한다.  The foreign recombinant production method preferably further comprises the step of culturing the mutant strain using a bioreactor, but is not limited thereto. In a specific embodiment of the present invention, the inventors have introduced a vector comprising a gene encoding glucose oxidase (GOD) or human serum albumin (HSA) to introduce HpGASl or GOD-producing strains and HSA-producing strains that produce foreign recombinant proteins. As a result of culturing the HpGAS2 gene and culturing, in particular, in the case of HpGASl gene disruption, the secretion ability of the foreign recombinant protein was significantly increased, and the activity of the produced protein was also increased (FIGS. 11, 12, and 13). Therefore, the Hanshenula polymorpha strain in which the HpGASl gene of the present invention is singly disrupted can be usefully used as a resource for the production of foreign recombinant proteins. In addition, the present invention provides a use of a Hansennura polymorpha (? / 7 «? / 3 polymorpha) variant strain that lacks the HpGASl gene as set forth in SEQ ID NO: 1 to improve protein secretion ability.
이하， 본 발명을 실시예 및 실험예에 의하여 상세히 설명한다. Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.
단， 하기 실시예 및 실험예는 본 발명을 구체적으로 예시하는 것일 뿐， 본 발명의 내용이 실시예 및 실험예에 의해 한정되는 것은 아니다.  However, the following Examples and Experimental Examples are only illustrative of the present invention in detail, and the content of the present invention is not limited by the Examples and Experimental Examples.
<실시예 1〉 HpGASl및 HpGAS2유전자 발굴 및 분석 Example 1 Discovery and Analysis of HpGASl and HpGAS2 Genes
본 발명자들은 한세눌라 폴리모르파 0 ^«¾//a polymorpha)^ 유전체 정보 해독 및 검색을 통하여 HpGASl유전자를 발굴하였다. The inventors have reported that the sensula polymorpha 0 ^ «¾ // a polymorpha) ^ genome information. HpGASl genes were identified through translation and search.
구체적으로， 미국 국립생물정보센터 (NCBI, http://丽 w.ncbi .nlm.nih.gov/)에서 다른 효모의 Gasl 단백질의 아미노산 서열정보 (PpGasl, ZbGasl, ScGasl 단백질)를 확보한 뒤， 본 발명자들이 구축한 한세눌라 폴리모르파 DL1 유전체 정보 데이터베이스에서 tblastn 프로그램을 통해 Gasl 단백질과 유사한 아미노산 서열을 갖는 단백질을 번역하는 5가지의 HpGAS 유전자 후보를 확보하였다. 그 증 상동성이 높은 두 유전자를 다른 효모 Gasl 단백질과 아미노산 서열의 상동성을 비교 및 선별하여 HpGASl 유전자 (서열번호 1) 및 ^β45 유전자 (서열번호 2)라고 명명하였다 (표 1). 그 후, 상기 HpGASl HpGAS2 유전자의 염기 서열을 BLAST로 확인한 결과， 신규한 유전자임을 알 수 있었다 (도 1 및 2). 또한， 상기 HpGASl 유전자의 아 :미노산 서열을 웹에서 이용 가능한 소프트웨어 (ClustalW-XXL ， http://www.ch.embnet.Org/software/ClustalW-XXLJi1:iTil)을 이용하여 이미 도메인 구조가 알려진 ScGasl의 아미노산 서열과 상동성을 분석하여 HpGasl 단백질에도 보존적인 아미노산서열 및 활성 도메인이 존재함을 확인하였다 (도 3). Specifically, after obtaining the amino acid sequence information (PpGasl, ZbGasl, ScGasl protein) of Gasl protein of other yeast from the National Institute of Biological Information (NCBI, http: // 丽 w.ncbi.nlm.nih.gov/), In the Hansennura polymorpha DL1 genome information database constructed by the present inventors, five HpGAS gene candidates were obtained through a tblastn program for translating proteins having amino acid sequences similar to those of Gasl proteins. The two highly homologous genes were compared and selected for homology between the other yeast Gasl protein and amino acid sequence and named HpGASl gene (SEQ ID NO: 1) and ^ β45 gene (SEQ ID NO: 2) (Table 1). Thereafter, as a result of confirming the nucleotide sequence of the HpGASl HpGAS2 gene by BLAST, it was found that it was a novel gene (FIGS. 1 and 2). In addition, ScGasl whose domain structure is already known using the software: ClustalW-XXL, http://www.ch.embnet.Org/software/ClustalW-XXLJi1:iTil, which is available on the web for the sub : minanoic acid sequence of the HpGASl gene. The homology with the amino acid sequence of confirmed that the conserved amino acid sequence and active domain also exist in the HpGasl protein (Fig. 3).
【표 1】 Table 1
identity if. polymorpha /. poiymorpha  identity if. polymorpha /. poiymorpha
C；,、 1 Cnsl Gasl Ga%1 G*\l  C ;, 1 Cnsl Gasl Ga% 1 G * \ l
Figure imgf000014_0001
<실시예 2> HpGASl및 HPGAS2단일 유전자 파쇄 균주 제작
Figure imgf000014_0001
Example 2 Preparation of HpGASl and H P GAS2 Single Gene Shredding Strains
<2-1> URA3파쇄 균주 제작  <2-1> URA3 crushed strain production
본 발명자들은 한세눌라 폴리모르파 균주의 醒 3 유전자를 파쇄하여 영양요구주로 만들기 위하여 URA3파쇄 카세트로 URA3유전자 절편 사이에 Zeocin 저항성 유전자 (ZeoR) 를 클로닝하였다. The present inventors cloned the Zeocin resistance gene (Zeo R ) between URA3 gene segments with the URA3 disruption cassette to disrupt the 醒 3 gene of Hanshenula polymorpha strain and make it a nutritional requirement.
구체적으로， 한세눌라 폴리모르파 DL1 균주로부터 분리한 염색체 DNA를 주형으로 하여 프라이머 ^ _N_Forward ( 열번호 3) 및 j¾i^?_N_Reverse_ZRF (서열번호' 4)를 이용하여 HpURA3^\ N-말단 절편 (499 bp)을 얻었으며， ^ —C— Forward— ZRR (서열번호 5) 및 ^ ^ C_Reverse (서열번호 6)를 이용하여 HpURA3^ Cᅳ말단 절편 (441 bp)을 중합효소 연쇄 반응 (PCR)을 통해 얻었다. 또한， pPICZalphaA 백터를 주형으로 하여 Zeo resistanceᅳ Forward (서열번호 7) 및 Zeo resistance_Reverse (서열번호 8)를 이용하여 Zeocin 저항성 유전자 (ZeoR) 카세트 (1192 bp)를 중합효소 연쇄 반응을 통해 얻었다. Fusion PCR 방법을 통해 상기 제작한 HpURA3^\ N-말단 절편， Zeocin 저항성 유전자 (ZeoR) 카세트 및 HpURA3≥ C-말단 절편을 이용하여 URA3 파쇄 카세트를 제작하였다. 猶3 파쇄 카세트를 야생형 한세눌라 폴리모르파 균주 polymorpha DLl), Glucose oxidase (GOD) 생산 재조합 균주 [ (？ r /?5/pDLM0X-G0D(H) ] 및 Human serum albumin (HSA)생산 재조합 균주 ( polymorpha G3T8)에 각각 상동재조합을 통해 도입하여 Zeocin 배지에서 생존가능한 URA3 유전자가 파쇄된 돌연변이 균주를 얻었다 (도 4). Specifically, a century Cronulla poly Maurepas to a chromosome DNA isolated from the DL1 strain as the template primer ^ _N_Forward (column No. 3) and j¾i ^? _ N_Reverse_ZRF using (SEQ ID NO: '4) HpURA3 ^ \ N- terminal fragment (499 bp), and HpURA3 ^ C ᅳ terminal fragment (441 bp) using ^ —C— Forward— ZRR (SEQ ID NO: 5) and ^ ^ C_Reverse (SEQ ID NO: 6) via polymerase chain reaction (PCR). Got it. In addition, the Zeocin resistance gene (Zeo R ) cassette (1192 bp) was obtained by polymerase chain reaction using Zeo resistance ᅳ Forward (SEQ ID NO: 7) and Zeo resistance_Reverse (SEQ ID NO: 8) using the pPICZalphaA vector as a template. The URA3 disruption cassette was prepared using the HpURA3 ^ \ N-terminal fragment, Zeocin resistance gene (Zeo R ) cassette, and HpURA3 ≧ C-terminal fragment prepared above by Fusion PCR.猶 3 crush cassette was used to produce wild type Hanshenula polymorpha DLl), Glucose oxidase (GOD) recombinant strain [(? R /? 5 / pDLM0X-G0D (H)] and human serum albumin (HSA) producing recombinant strain ( polymorpha G3T8) was introduced through homologous recombination, respectively, to obtain mutant strains in which the viable URA3 gene was disrupted in Zeocin medium (FIG. 4).
<2-2> HpGASl파쇄 균주 제작 <2-2> HpGASl crushed strain preparation
본 발명자들은 HpGASl 유전자가 결손된 파쇄 균주를 제작하기 위하여 GAS1 파쇄 카세 "트를 제작하여 U 3 유전자가 파쇄된 실시예 <2-1〉의 돌연변이 균주에 도입하였다.  The present inventors prepared a GAS1 disruption cassette "to generate a disrupted strain lacking the HpGASl gene and introduced it into the mutant strain of Example <2-1> in which the U 3 gene was disrupted.
구체적으로， 한세눌라 폴리모르파 DL1 균주로부터 분리한 염색체 DNA를 주형으로 하여 ^^45_/_N_Forward (서열번호 9) 및 v¾o^5_N_Reverse_UJF (서열번호 10)를 이용하여 HpGASl^\ N-말단 절편 (537 bp)을 얻었으며， ^^5 _CJ rward— LUR (서열번호 11) 및 ^ A _C— Reverse (서열번호 12)를 이용하여 HpGASlS\ C-말단절편 (524 bp)을 중합효소 연쇄반웅 (PCR)을 통해 얻었다. pLaclIR3를 주형 (Kim et al. , J Biol Chem. 281, 6261, 2006)으로 프라이머 쌍 Hp 1 acZ_URA3_N_Forward (서열번호 24)와 HplacZ_URA3_N_Reverse (서열번호 25)를 이용하여 ᅳ lacZ-HPURA3-lacZ 카세트의 N—말단 절편을， HplacZ_URA3_C_Forward (서열번호 26)와 Hp lacZ_URA3_C— Reverse (서열번호 27)를 이용하여 lacZ-HPU A3-lacZ 카세트의 C-말단 절편을 증합효소 연쇄반응을 통해 얻었다. HpGASlS\ N-말단절편， lacZ-HpURA3-lacZ 카세트의 N-말단 절편을 fusion PCR을 통해 GAS1 파쇄 카세트 _N -말단 절편을 얻고， lacZ-HpURA3-lacZ 카세트의 C—말단 절편， HpGASl^ C-말단절편을 fusion PCR을 통해 GAS1 파쇄 카세트 _Cᅳ말단 절편을 얻었다 (도 5). 제작한 GAS1 파쇄 카세트ᅳ N-말단 절편과 GAS1 파쇄 카세트 _C-말단 절편을 상기 실시예 <2-1>에서 제작한 URA3 유전자가 파쇄된 균주 각각에 상동재조합을 통해 도입하여 SC-URA 최소 배지에서 생존할 수 있는 HpGASl유전자가 파쇄된 돌연변이주를 얻었다. Specifically, ^^ 45 _ / _ N_Forward (SEQ ID NO: 9) and v¾o ^ 5_N_Reverse_UJF (SEQ ID NO: 9) were used as chromosomal DNA isolated from Hanshenula polymorpha DL1 strain. 10) was used to obtain HpGASl ^ \ N-terminal fragment (537 bp), and ^ p5SlS \ C- using ^^ 5 _CJ rward— LUR (SEQ ID NO: 11) and ^ A _C— Reverse (SEQ ID NO: 12). Terminal fragments (524 bp) were obtained via polymerase chain reaction (PCR). pLaclIR3 as a template (Kim et al., J Biol Chem. 281, 6261, 2006) using primer pairs Hp 1 acZ_URA3_N_Forward (SEQ ID NO: 24) and HplacZ_URA3_N_Reverse (SEQ ID NO: 25) for the expression of lacZ-H P URA3-lacZ cassette. N-terminal fragments were obtained by HplacZ_URA3_C_Forward (SEQ ID NO: 26) and Hp lacZ_URA3_C—Reverse (SEQ ID NO: 27) to obtain C-terminal fragments of the lacZ-H P U A3-lacZ cassette by polymerase chain reaction. HpGASlS \ N-terminal fragment, N-terminal fragment of lacZ-HpURA3-lacZ cassette, were obtained by GAS1 disruption cassette _N-terminal fragment by fusion PCR, C-terminal fragment of lacZ-HpURA3-lacZ cassette, HpGASl ^ C-terminal Sections were obtained by GAS1 disruption cassette _C ᅳ terminal fragments by fusion PCR (Fig. 5). The prepared GAS1 disrupted cassette ᅳ N-terminal fragment and the GAS1 disrupted cassette _C-terminal fragment were introduced into each of the strains of the URA3 gene fragmented in Example <2-1> by homologous recombination, and then, in the SC-URA minimal medium. A mutant strain in which the viable HpGASl gene was disrupted was obtained.
<2-3> HpGAS2파쇄 균주 제작 <2-3> HpGAS2 crushed strain preparation
본 발명자들은 HPGAS2유전자가 결손된 파쇄 균주를 제작하기 위하여 파쇄 카세트를 제작하여 실시예 <2-1>의 UM3 유전자가 파쇄된 돌연변이주에 도입하였다. The inventors prepared a disruption cassette to introduce a mutant strain in which the UM3 gene of Example <2-1> was disrupted to prepare a disrupted strain lacking the H P GAS2 gene.
구체적으로， 한세눌라 폴리모르파 Ll 균주로부터 분리한 염색체 DNA를 주형으로 하여 ^fi5_N_Forward (서열번호 13) 및 ^i S?_N_Reverse_LUF (서열번호 14)를 이용하여 HPGAS2S N-말단 절편 (537 bp)을 얻었으며， ^ί 5ᅳ C_Forward_LU (서열번호 15) 및 ^fi!S2 _Reverse (서열번호 16)를 이용하여 HPGAS22 C-말단절편 (524 bp)을 중합효소 연쇄반웅 (PCR)을 통해 얻었다. pLacUR3를 주형으로 프라이머 쌍 HplacZ_URA3_N_Forward (서열번호 24)와 HplacZ— URA3_N_Reverse (서열번호 25)를 이용하여 lacZ— HpU A3-lacZ 카세트의 N-말단 절편을， HplacZ_URA3_C_Forward (서열번호 26)와 HplacZ_URA3_C_Reverse (서열번호 27)를 이용하여 lacZ-HpURA3-lacZ 카세트의 C-말단 절편을 중합효소 연쇄반웅을 통해 얻었다. HpGAS2^\ Nᅳ말단절편, lacZ-HpURA3-lacZ 카세트의 Nᅳ말단 절편을 fusion PCR을 통해
Figure imgf000017_0001
파쇄 카세트ᅳ Nᅳ말단 절편을 얻고， lacZ-HpURA3-lacZ 카세트의 Cᅳ말단 절편, HPGAS2 C-말단절편을 fusion PCR을 통해 G S^파쇄 카세트 _C-말단 절편을 얻었다 (도 6). 제작한 파쇄 카세트ᅳ Ν-말단 절편과 GAS2 파쇄 카세트 _C-말단 절편을 상기 실시예 <2— 1>에서 제작한 iK4 유전자가 파쇄된 균주 각각에 상동재조합을 통해 도입하여 SC-URA 최소 배지에서 생존할 수 있는 HPGAS2 유전자가 파쇄된 돌연변이주를 얻었다. Specifically, H P GAS2S N-terminal fragment (537 bp) using ^ fi5_N_Forward (SEQ ID NO: 13) and ^ i S? _N_Reverse_LUF (SEQ ID NO: 14) using chromosomal DNA isolated from Hanshenula polymorph L1 strain as a template. ), And HPGAS2 2 C-terminal fragment (524 bp) was obtained through polymerase chain reaction (PCR) using ^ ί 5 ᅳ C_Forward_LU (SEQ ID NO: 15) and ^ fi! S2 _Reverse (SEQ ID NO: 16). . Using the pLacUR3 template, the N-terminal fragment of the lacZ—HpU A3-lacZ cassette was prepared using the primer pairs HplacZ_URA3_N_Forward (SEQ ID NO: 24) and HplacZ—URA3_N_Reverse (SEQ ID NO: 25). C-terminal fragments of the lacZ-HpURA3-lacZ cassette were obtained by polymerase chain reaction using HplacZ_URA3_C_Forward (SEQ ID NO: 26) and HplacZ_URA3_C_Reverse (SEQ ID NO: 27). HpGAS2 ^ \ N ᅳ fragment and N ᅳ terminal fragment of lacZ-HpURA3-lacZ cassette were subjected to fusion PCR.
Figure imgf000017_0001
N-terminal fragments were obtained from the shredding cassette ，, C ^ -terminal fragments of the lacZ-HpURA3-lacZ cassette, and H P GAS2 C-terminal fragments were obtained from the GS ^ crushing cassette_C-terminal fragments by fusion PCR (FIG. 6). The resulting fragmented cassette ᅳ Ν-terminal fragment and the GAS2 fragmented cassette _C-terminal fragment were introduced into each of the strains of the iK4 gene fragmented in Example <2-1> through homologous recombination to survive in the SC-URA minimal medium. Mutant strains in which the H P GAS2 gene was disrupted were obtained.
<실험예 1>유전자 파쇄 변이주의 특성 확인 <Experimental Example 1> Confirmation of the characteristics of the gene disruption mutation strain
<1ᅳ 1> ¾?ί 5 또는 7¾?0 "파쇄 균주의 형태학적 특징 확인  <1 ᅳ 1> Morphological characterization of ¾? Ί 5 or 7¾? 0 "crushed strains
본 발명자들은 상기 <실시예 2>에서 제작한 HpGASl 유전자 파쇄 균주 및 τ¾?ί 5 유전자 파쇄 균주의 형태적 특징을 확인하기 위하여 공초점 현미경과 광학 현미경으로 한세눌라 폴리모르파 야생형 DL1 균주와 비교하였다.  The present inventors compared the Hansenula polymorpha wild type DL1 strain with confocal microscopy and optical microscope to confirm the morphological characteristics of the HpGASl gene disruption strain and τ¾ ί 5 gene disruption strain prepared in <Example 2> .
구체적으로, 한세눌라 폴리모르파 야생형 DL1균주， ？^ ^유전자 파쇄 균주 및 HpGAS2유전자 파쇄 균주들을 각각 3 ml 포도당 2% 복합 배지에 200 rpm으로 Specifically, Hanshenula polymorpha wild type DL1 strain,? ^ ^ Gene disruption strain and HpGAS2 gene disruption strains, respectively, at 200 rpm in 3 ml glucose 2% complex medium
370C에서 16시간 동안 전 배양한 후 초기 0D600 값이 0.3이 되도록 50 ml 포도당 2% 복합 배자와 포도당 2% 최소 배지에 접종한 후 같은 조건으로 본 배양하였다. 48시간 뒤 정체기의 세포 배양액을 0D600 값이 10이 되도록 취한 후 13,000 rpm으로After incubation for 16 hours at 37 0 C inoculated in 50 ml glucose 2% complex embryo and glucose 2% minimum medium to the initial 0D 600 value 0.3 and then incubated under the same conditions. After 48 hours, the cell culture medium in the standing phase was taken to have a value of 0D 600 of 10, and then at 13,000 rpm.
10분 동안 40C에서 원심분리한 후 상등액을 제거하고 세포를 얻었다. 상기 세포를 lxPBS로 두 번 세척한후 칼코플로어 화이트 (Calcofluor White, CFW) 용액 (1 mg/ml) 100 ^로 15분 동안 상온에서 반응시켰다. 반응 후 lxPBS로 두 번 세척하고 lxPBS 100 !ii — 세포를 푼 뒤 슬라이드에 10 ^을 올려 Excitation 405 nm, Emission 420-480 nm에서 2000 배율의 공초점 현미경 및 1000 배율의 광학 현미경을 이용하여 관찰하였다 (도 8). 그 결과， HpGASl 유전자 파쇄 균주는 세포의 형태가 원형이 되었고 야생형보다 그 크기가 두 배정도 커진 것이 관찰되었으며， 세포들이 다소 덜 분리되고 서로 뭉쳐있는 것을 확인할 수 있었다. 반면，
Figure imgf000018_0001
유전자 파쇄 균주는 야생형과 비슷한 모양을 보였다. After centrifugation at 4 0 C for 10 minutes, the supernatant was removed and cells were obtained. The cells were washed twice with lxPBS and then reacted with Calcofluor White (CFW) solution (1 mg / ml) at 100 ^ for 15 minutes at room temperature. After the reaction, the cells were washed twice with lxPBS and lxPBS 100! Ii — cells were unloaded and 10 ^ was placed on the slides and observed by using a confocal microscope at 2000 magnification and an optical microscope at 1000 magnification at Excitation 405 nm and emission 420-480 nm. (FIG. 8). As a result, the HpGASl gene disruption strain was found to be circular in cell form and twice as large as the wild type. I could see that they were separated and stuck together. On the other hand,
Figure imgf000018_0001
The gene disruption strains were similar to wild type.
<1-2> HpGASl또는 HpGAS2파쇄 균주의 생리적 특성 확인 <1-2> Confirmation of Physiological Characteristics of HpGASl or HpGAS2 Fracture Strains
본 발명자들은 상기 <실시예 2>에서 제작한 HpGASl 유전자 파쇄 균주 및 ^5 유전자 파쇄 균주의 생리적 특징을 세포벽 저해 물질에 대한 감수성을 통해 확인하였다.  The present inventors confirmed the physiological characteristics of the HpGASl gene disruption strain and ^ 5 gene disruption strain prepared in <Example 2> through the sensitivity to the cell wall inhibitor.
구체적으로, HpGASl 유전자 파쇄 또는 HPGAS2 유전자 파쇄에 의해 변화된 세포벽의 구조에 따른 세포벽 합성저해제 및 삼투교란제에 대한 감수성을 확인하기 위하여， 한세눌라 폴리모르파 DL1 야생형 균주， HpGASl 파쇄 균주 및 HpGAS2파쇄 균주들을 각각 3 ml 포도당 2% 복합 배지에 200 rpm으로 37°C에서 16시간 동안 전 배양하였다. 상기 배양한 균주들을 멸균수 1 ml에 처음 0D6O0 값을 1로 시작하여 연속적으로 1/10씩 회석하여 포도당 2% 복합 한천배지, 세포벽 합성 저해제인 3% 칼코플로어 화이트 (Calcofluor White, CFW) , 1% 콩고레드 (Congo Red, C ) 및 삼투교란제인 0.01%도데실 황산나트륨 (Sodium Dodecyl Sulfate, SDS)이 각각포함된 포도당 2% 복합 한천 배지에 1 씩 집적하여 37°C에서 이를 또는 삼일 동안 배양하였고, 아울러， 열 스트레스에 대한 감수성을 확인하기 위해 포도당 2% 복합 한천배지에 집적하여 42°C에서 이를 동안 배양하였다. 그 결과, 세포벽의 구조 변화로 인해 HpGASl 유전자 파쇄 균주는 대조군에 비해 37°C와 420C 포도당 2% 복합 한천배지에서 생장이 저해되었으며， 세포벽 합성 저해제 감수성 배지 및 삼투교란제 감수성 배지에서도 야생형 또는 HPGAS2 파쇄 균주에 비해 생장이 저해되고 성장속도가 느려짐을 확인할 수 있었다 (도 9의 1, 2 및 3). <실험예 >유전자 파쇄 변이주의 총 단백질 분비능 확인 Specifically, in order to confirm susceptibility to cell wall synthesis inhibitors and osmotic disturbances according to the structure of the cell wall changed by HpGASl gene disruption or H P GAS2 gene disruption, Hansenula polymorpha DL1 wild type strain, HpGASl fragmented strain and HpGAS2 fragmented. The strains were preincubated for 16 hours at 37 ° C at 200 rpm in 3 ml glucose 2% complex media each. The cultured strains were first diluted with 1/10 of 1D in 1 ml of sterile water, and then serialized 1/10 for glucose 2% complex agar medium, 3% Calcofluor White (CFW), a cell wall synthesis inhibitor, 1% Congo Red (C) and osmotic perturbant 0.01% sodium dodecyl sulfate (SDS), respectively, were collected in a glucose 2% complex agar medium containing 1% and incubated at 37 ° C for 3 days. In addition, in order to confirm the susceptibility to heat stress was integrated in glucose 2% complex agar medium and cultured at 42 ° C during this. As a result, HpGASl gene disruption strains were inhibited in 37% C and 42 0 C glucose 2% agar medium due to the change of cell wall structure, and wild type or even in cell wall synthesis inhibitor-sensitive media and osmotic disturbance-sensitive media. It was confirmed that growth was inhibited and growth rate was slow compared to H P GAS2 crushed strains (FIGS. 1, 2 and 3 of FIG. 9). Experimental Example Confirmation of Total Protein Secretion Ability of Gene Breaking Mutant
본 발명자들은 HpGASl 유전자 파쇄 및 HpGAS2유전자 파쇄에 따른 세포벽의 구조 변화에 의해 총 단백질 분비량이 증가하는지 확인하기 위하여 브래드포드 분석 (Bradford assay) 방법으로 확인하였다. The inventors of the present invention have found that the cell wall following HpGASl gene disruption and HpGAS2 gene disruption is In order to confirm whether the total protein secretion is increased by the structural change was confirmed by the Bradford assay method.
구체적으로， 한세눌라 폴리모르파 DL1야생형 균주， ;¾^A5 유전자 파쇄 균주 및 HPGAS2유전자 파쇄 균주를 3 ml 포도당 2Ά> 복합 배지에 200 rpm으로 37°C에서 16시간 동안 전 배양한 후 50 ml 포도당 2% 복합 배지에 초기 0D600 값이 0.3이 되도록 접종하여 같은 조건으로 본 배양하였다. 배양 시간대 별로 배양액을 취하여Specifically, Hanshenula polymorpha DL1 wild type strain,; ¾ ^ A5 gene disruption strain and H P GAS2 gene disruption strain was pre-incubated for 16 hours at 37 ° C at 200 rpm in 3 ml glucose 2> complex medium 50 Incubated in ml glucose 2% complex medium to the initial 0D 600 value 0.3 to the same conditions. Take culture medium at each time of incubation
13,000 rpm으로 10분 동안 4°C에서 원심분리한 후 상등액을 확보하여 분비된 단백질 양을 브래드포드 (protein assay dye reagent concentrate -BI0RAD) 분석 방법으로 측정하였으며， bovine serum albumin (BSA)을 사용하여 스탠다드 커브를 그리고 측정된 단백질 양을 0D600값으로 노말라이제이션 하였다. 그 결과, 야생형에 비하여 HpGASl 유전자 파쇄 균주에서 시간 의존적으로 총 단백질 분비량이 늘어남을 확인하였고， 지수기의 후기를 넘어 정체기가 지날수록 총 분비된 .단백질의 양이 확연히 차이가 나는 것을 확인하였다 (도 10). ¾^AS?유전자 파쇄 균주는 야생형과 비슷한 단백질 분비량이 확인되었다. : After centrifugation at 4 ° C for 10 minutes at 13,000 rpm, the supernatant was secured and the amount of secreted protein was measured by Bradford (protein assay dye reagent concentrate -BI0RAD) method, and standardized using bovine serum albumin (BSA). The curve was drawn and the measured protein amount normalized to 0D 600 value. As a result, it was confirmed that the total protein secretion increased in time-dependent manner in HpGASl gene disrupted strains compared to wild type, and that the total amount of protein secreted. 10). Gene fragmentation similar to wild type was confirmed in the ¾ ^ AS gene disruption strain. :
<실험예 3>유전자 파쇄 변이주의 외래 재조합 단백질 분비능 확인 Experimental Example 3 Confirmation of Foreign Recombinant Protein Secretion Ability
<3-1> GOD 생산균주의 Glucose oxidase 분비능 확인  <3-1> Glucose oxidase secretion ability of GOD producing strain
본 발명자들은 HpGASl 유전자 파쇄 HpGAS2 유전자 파쇄에 의해 GOD 생산균주의 Glucose oxidase의 분비량이 증가하는지 확인하였다.  The present inventors confirmed whether the secretion of Glucose oxidase from the GOD producing strain was increased by HpGASl gene disruption.
구체적으로， 야생형 GOD 생산균주 [H. poIymorpha/pIAMOX-GOO )] , HpGASl 유전자 파쇄 균주 [ polymorpha AffpGASl/pOLV X-GOm)] 및 HpGAS2 유전자 파쇄 균주 [ . polymorpha o^5 /pDLM0X-G0D(H)]를 3 ml 포도당 2% 복합 배지에 200 rpm으로 37°C에서 16시간 동안 전 배양한 후， M0X프로모터를 이용해 발현되는 해당 외래 재조합 단백질의 발현유도를 위해 메탄을 2%복합 배지 50 ml에 초기 0D600값이 0.3이 되도록 접종한 후 같은 조건으로 본 배양하였다. 그 후， 0D600값 2에 해당하는 배양액을 취한 후 13,000 rpm으로 10분 동안 4°C에서 원심분리하여 상등액을 확보하였다. 상기 상등액을 5x샘플 로딩 버퍼와 흔합하여 5분 동안 끓인 후 샘플을 절반씩 나눠서 (즉, 0D600 값 1에 해당하는 상등액) 8¾> SDS-PAGE 젤 두 조에 전기영동하였다. 젤 두 조 중 하나는 Coomassie Brilliant Blue R-250 Staining Solution(BIO-RAD)으로 염색하고 destaining 버퍼로 탈색하였다. 다른 젤은 PVDF 막으로 단백질을 옮긴 후 해당 PVDF 막을 5%스킴 밀크 용액으로 차단하였으며， 1차 항체인 His-probe(Santa Cruz Biotechnology)를 1:1000으로 희석하여 2시간 동안 반웅시켰다. 반웅 후， TBST(50 mM Tris-HCKpH 7.5)， 150 mM NaCl 및 0.05¾> Tween 20) 용액으로 10분씩 3번 씻은 뒤 2차 항체인 Anti-Mouse IgG(Sigma)를 1:3000으로 희석하여 1시간 30분 동안 반웅시켰다. 그 후， TBST 용액으로 10분씩 3번 씻어준 뒤 ECL advanceTM Western Blotting Detect ion Ki t (AmershamTM-GE Healthcare)으로 검출하였디-. Specifically, wild type GOD producing strain [H. poIymorpha / pIAMOX-GOO)], HpGASl gene disruption strain [polymorpha AffpGASl / pOLV X-GOm)] and HpGAS2 gene disruption strain [. polymorpha o ^ 5 / pDLM0X-G0D (H)] was incubated in 3 ml glucose 2% complex medium at 200 rpm for 16 hours at 37 ° C, and then the expression of the foreign recombinant protein expressed using the M0X promoter was determined. Hazardous methane was inoculated in 50 ml of 2% complex medium to give an initial 0D 600 value of 0.3, followed by main culture. After that, 0D 600 corresponds to 2 After taking the culture solution, the supernatant was secured by centrifugation at 4 ° C for 10 minutes at 13,000 rpm. The supernatant was mixed with 5 × sample loading buffer, boiled for 5 minutes, and then electrophoresed in two pairs of 8¾> SDS-PAGE gels by dividing the sample in half (ie, the supernatant corresponding to 0D 600 value 1). One of the two gels was stained with Coomassie Brilliant Blue R-250 Staining Solution (BIO-RAD) and bleached with destaining buffer. The other gel was transferred to a PVDF membrane, and then the PVDF membrane was blocked with 5% skim milk solution, and the primary antibody His-probe (Santa Cruz Biotechnology) was diluted 1: 1000 and reacted for 2 hours. After reaction, wash three times for 10 minutes with TBST (50 mM Tris-HCKpH 7.5), 150 mM NaCl and 0.05¾> Tween 20), and then dilute the secondary antibody Anti-Mouse IgG (Sigma) to 1: 3000. The reaction was carried out for 30 minutes. Then, washed with TBST solution three times for 10 minutes and detected by ECL advanceTM Western Blotting Detect ion Kit (AmershamTM-GE Healthcare).
그 결과， 염색한 젤에서는 전체적으로 HpGASl 파쇄 균주의 샘플에서 단백질 밴드가 진하게 나와서 총 단백질이 많이 분비된 것을 확인할 수 있었으며, 웨스턴 블랏의 결과를 통해 100 KDa 부근에 Glucose oxidase가 발현된 것을 확인할 수 있었고 야생형과 HpGAS2 파쇄 균주보다 특히 HpGASl 파쇄 균주의 샘플에서 많은 양의 GOD가 검출 되었다 (도 11).  As a result, in the stained gels, the protein bands of the HpGASl crushed strains were concentrated in total, and the total protein was secreted, and the Western blot showed that Glucose oxidase was expressed near 100 KDa. A greater amount of GOD was detected in the samples of the HpGASl disrupted strains than in the HpGAS2 disrupted strains (FIG. 11).
또한， 배양 상등액으로 분비된 Glucose oxidase의 활성을 GLUCOSE OXIDASE 분석 키트 (MEGAZYME)로 측정한 결과， 야생형에 비해 HpGASl 파쇄 균주의 배양 상등액에서 1.9배 증가함을 확인하였다 (도 12). 따라서 야생형 균주에 비하여 HpGASl파쇄 균주에서 GOD분비량이 늘어날 뿐만 아니라 활성형태로 분비가 잘 됨을 확인하였다. ^ A 파쇄 균주는 야생형과 비슷한 활성측정 값을 보였다.  In addition, the activity of the glucose oxidase secreted into the culture supernatant was measured by GLUCOSE OXIDASE assay kit (MEGAZYME), it was confirmed that 1.9-fold increase in the culture supernatant of HpGASl crushed strain compared to wild type (Fig. 12). Therefore, as well as increased GOD secretion in HpGASl crushed strain compared to wild-type strain, it was confirmed that the secretion in the active form is well. ^ A disrupted strain showed similar activity measurement values as wild type.
<3-2> G3T8 균주의 사람 혈청 알부민 (Human serum albumin) 분비능 확인 본 발명자들은 HpGASl 유전자 파쇄 및 HpGAS2 유전자 파쇄에 의해 재조합 인간 혈청 알부민 (Human serum albumin, HSA) 생산균주의 HSA분비량이 증가하는지 확인하였다. ' 구체적으로 , 야생형 HSA 생산균주 ( polyniorpha G3T8) , HpGASl유전자 파쇄 균주 ( . polyniorpha G3T8 AHpGASl) 및 HpGAS2유전자 파쇄 균주 07. polyniorpha G3T8 ᅀ HPGAS2)^ 3 ml 포도당 2% 복합 배지에 200 rpm으로 37°C에서 16시간 동안 전 배양한 후 50 ml 메탄올 2% 복합 배지에 초기 0D600 값이 0.3이 되도록 접종한 후 같은 조건으로 본 배양한 후， 이때 HSA 발현을 유도하기 위하여 메탄올 2% 복합 배지를 사용하여 배양하였다. 그 후， 0D600 값이 0.1이 되도록 배양액을 취한 후<3-2> Confirmation of secretion of human serum albumin of G3T8 strain The present inventors confirmed whether HSA secretion of recombinant human serum albumin (HSA) producing strain was increased by HpGASl and HpGAS2 gene disruption. It was. ' Specifically, wild type HSA producing strain (polyniorpha G3T8), HpGASl gene disruption strain (.polyniorpha G3T8 AHpGASl) and HpGAS2 gene disruption strain 07. polyniorpha G3T8 ᅀ H P GAS2) ^ 3 ml glucose 2% at 37 rpm at 200 rpm After incubation for 16 hours in C, 50 ml methanol 2% complex medium was inoculated so that the initial 0D 600 value was 0.3, followed by main culture under the same conditions, and then methanol 2% complex medium was used to induce HSA expression. And incubated. After that, the culture medium is taken so that the value of 0D 600 becomes 0.1.
13,000 rpm으로 10분 동안 4°C에서 원심분리하여 상등액을 확보하였다. 상기 상등액을 5x샘플 로딩 버퍼와 흔합하여 5분 안 끓인 후 샘플을 절반씩 나눠서 (즉， 0D600값 0.05에 해당하는 상등액) 1OT SDS— PAGfe젤 두 조에 전기영동하였다. 젤 두 조 중 하나는 Coomassie Brilliant Blue R_250 Staining Solution(BIO-RAD)으로 염색하고 destaining 버퍼로 탈색하였다. 다른 젤 하나는 단백질을 PVDF 막으로 옮긴 후 해당 PVDF 막을 5% 스킴 밀크 용액으로 차단하였으며， 1차 항체인 Anti-Human Albumin antibody(Sigma)를 1:10000으로 희석하여 2시간 동안 반웅시켰다. 반웅 후, TBST(50 mM Tris-HCKpH 7.5), 150 mM NaCl 및 0.05% Tween 20) 용액으로 10분씩 3번 씻은 뒤 2차 항체인 goat ant i -rabbit IgG-HRP(Santa Cruz Biotechnology)를 1:5000으로 희석하여 1시간 30분 동안 반웅시켰다. 그 후, TBST용액으로 10분씩 3번 씻어준 뒤 ECL Western Blotting Detection Kit (AmershamTM-GE Healthcare)으로 검출하였다. 그 결과, 염색한 젤에서는 전체적으로 HpGASl 파쇄 균주의 샘플에서 단백질 밴드가 진하게ᅵ 나와 총 단백질이 많이 분비된 것을 확인할 수 있었으며, 웨스턴 블랏의 결과를 통해 66 Da 부근에 인간 혈청 알부민이 검출된 것을 확인할 수 있었고 HpGASl 파쇄 균주의 샘플에서 야생형과 HpGAS2 파쇄 균주보다 많은 양의 HSA가 검출 되었다 (도 13). The supernatant was obtained by centrifugation at 4 ° C. for 10 minutes at 13,000 rpm. The supernatant was mixed with 5 × sample loading buffer and boiled for less than 5 minutes, and then electrophoresed in two pairs of 1OT SDS—PAGfe gels by dividing the samples in half (ie, the supernatant corresponding to a 0D 600 value of 0.05). One of the two gels was stained with Coomassie Brilliant Blue R_ 2 50 Staining Solution (BIO-RAD) and bleached with destaining buffer. The other gel was transferred to a PVDF membrane, and the PVDF membrane was blocked with a 5% skim milk solution, and the first antibody, Anti-Human Albumin antibody (Sigma), was diluted 1: 10000 and reacted for 2 hours. After reaction, washed three times for 10 minutes with TBST (50 mM Tris-HCKpH 7.5), 150 mM NaCl and 0.05% Tween 20) solution, and then the second antibody, goat ant i -rabbit IgG-HRP (Santa Cruz Biotechnology) 1: Diluted to 5000 and reacted for 1 hour 30 minutes. Thereafter, the mixture was washed three times with TBST for 10 minutes, and then detected by ECL Western Blotting Detection Kit (AmershamTM-GE Healthcare). As a result, in the stained gel, the protein band of the HpGASl crushed strain was found to be high, and the total protein was secreted. The result of Western blot showed that human serum albumin was detected near 66 Da. In the samples of HpGASl crushed strains, more HSA was detected than wild type and HpGAS2 crushed strains (FIG. 13).
<실험예 4>유전자 회복에 의한 기능 보완 확인 Experimental Example 4 Functional Complement Confirmation by Gene Recovery
<4-1> HpGASl및 HpGAS2유전자 파쇄 균주의 腿3파쇄  <4-1> 腿 3 crushing of HpGASl and HpGAS2 gene disruption strains
본 발명자들은 상기 실험예와 같은 효과가 lacZ-URA3ᅳ lacZ카세트에 의해 HPGAS17\ 파쇄되었기 때문인지 확인하기 위하여 유전자 희복에 의한 기능 보완 실험을 하고자 하여, 상기 유전자 파쇄 균주에서 lacZᅳ URA3-lacZ 카세트를 제거하였다. The present inventors have the same effect as the above experimental example by lacZ-URA3 ᅳ lacZ cassette In order to perform a function complement experiment by gene leaning to determine whether H P GAS17 \ was disrupted, the lacZ ᅳ URA3-lacZ cassette was removed from the gene disruption strain.
구체적으로， lacZ-URA3-lacZ 제거용 카세트를 제작하기 위하여 한세눌라 폴리모르파 DL1 균주로부터 분리한 염색체 DNA를 주형으로 ^ AWJLForward (서열번호 9) 및 ^ A _N—Reverse (서열번호 10)를 이용하여 HpGASl^ N-말단 절편 (537 bp)을， ¾ 247_C_Forward_GNR (서열번호 23)및 ¾? A _C— Reverse (서열번호 12)를 이용하여 HpGASl^ N-말단 절편과 융합할 수 있는 HPGAS1^\ C—말단 절편 (524 • bp)을 중합효소 연쇄 반응 (PCR)을 -통해 얻었다. 상기 두 절편을 fusion PCR하여 lacZ-URA3-lacZ 제거용 카세트를 얻은 후 HPGAS1<A 파쇄된 균주에 상동재조합을 통해 도입하여 醒3유전자가 없는 균주만 생장 가능한 5-F0A 배지에서 생존가능한Specifically, chromosomal DNA isolated from Hanshenula polymorpha DL1 strain was used as a template to prepare lacZ-URA3-lacZ removal cassette using ^ AWJLForward (SEQ ID NO: 9) and ^ A _N—Reverse (SEQ ID NO: 10). HpGASl ^ N-terminal fragment (537 bp), ¾ 247_C_Forward_GNR (SEQ ID NO: 23) and ¾? H P GAS1 ^ \ C-terminal fragments (524 • bp) that could be fused with HpGASl ^ N-terminal fragments using A_C—Reverse (SEQ ID NO: 12) were obtained via polymerase chain reaction (PCR). After fusion PCR of the two fragments, a cassette for removing lacZ-URA3-lacZ was obtained, followed by homologous recombination into H P GAS1 <A crushed strains, and thus viable in 5-F0A medium capable of growing only strains without the 醒 3 gene.
URA3유전자가 파쇄된 돌연변이주 ( . polyinorpha DL1 AHpGASl ΔΙ Α3)를 얻었다 (도 A mutant strain (.polyinorpha DL1 AHpGASl ΔΙ Α3) with URA3 gene disrupted was obtained (Fig.
14). <4-2> HpGASl, HpGAS2및 ScGASl유전자 도입 14). <4-2> Introduction of HpGASl, HpGAS2 and ScGASl Genes
- 본 발명자들은 HpGASl, HpGAS2 및 ScGASl 유전자가 각각 클로닝된 백터를 제작하여 상기 HpGASl유전자 파쇄 균주 ( . polyinorpha DL1 AHpGASl ΐίί ?)에 각각 도입하였다.  The present inventors produced vectors in which the HpGASl, HpGAS2 and ScGASl genes were cloned, respectively, and introduced them into the HpGASl gene disruption strain (.polyinorpha DL1 AHpGASl ΐίί?).
구체적으로， 한세눌라 폴리모르파 DL1 균주로부터 분리한 염색체 DNA를 주형으로 각각 서열번호 17및 18，서열번호 19및 20의 프라이머를 이용하여 증폭한 HpGASl, HpGAS2 유전자 카세트와 사카로마이세스 세레비지애 BY4741 균주로부터 분리한 염색체 DNA를 주형으로 서열변호 21 및 22의 프라이머를 이용하여 증폭한 Specifically, HpGASl, HpGAS2 gene cassette and Saccharomyces cerevisiae amplified using primers of SEQ ID NOs: 17 and 18, and SEQ ID NOs: 19 and 20, respectively, as a template of chromosomal DNA isolated from Hanshenula polymorpha DL1 strain. Chromosomal DNA isolated from BY4741 strain was amplified using primers of SEQ ID NOs: 21 and 22 as templates.
ScGASl 유전자 카세트를, 제한효소 _ MssJ을 처리한 pDLGUK 백터에 평활 말단으로 클로닝 하였다 (도 7의 1). pDLGUK 백터는 염색체 DNA의 텔로미어 부근으로 무작위적으로 삽입할 수 있는 鹏및 ^i?유전자를 가진 백터로써， 형질전환체는 iffi ?유전자가 삽입되기 때문에 SC-URA 최소배지에서 자라는 형질전환체를 얻을 수 있다. 상기 HpGASrA 삽입된 백터 (pDLGUK-y¾^7A«), HpGAS27\ 삽입된 백터 ( MM-HpGAS2)， 쑈 가：삽입된 백터 ( pDLGUK- ScGASl) 및 pDLGUK 공백터를 URA3유전자 및 HPGAS17\ 파쇄된 한세눌라 폴리모르파 DL1 균주 ( . polymorpha DL1 ᅀ HpGASl Δ羅 3)쒜 각각 형질전환하였다. The ScGASl gene cassette was cloned into the blunt ends in the pDLGUK vector treated with restriction enzyme _ MssJ (FIG. 1). The pDLGUK vector is a vector with genes ^ and ^ i? that can be randomly inserted near the telomeres of chromosomal DNA. The transformant is capable of obtaining transformants that grow on SC-URA minimal media because the iffi gene is inserted. Number have. The HpGASrA inserted vector (pDLGUK-y¾ ^ 7A «), HpGAS27 \ inserted vector (MM-HpGAS2), 쑈 A: inserted vector (pDLGUK- ScGASl) and pDLGUK blank were URA3 gene and H P GAS17 \ crushed Hansenula polymorpha DL1 strain (.polymorpha DL1 ᅀ HpGAS Δ 羅 3) was transformed.
그 결과， SC-URA 최소 배지에서 생장: 가능한 백터가 삽입된 형질전환체 H. polymorpha DL1 AHpGASl / ¾ ?/pDLGUKᅳ HpGASl， H. polymorpha DL1 AHpGASl l^4.?/pDLGUK-HpGAS2, H. polymorpha DL1 AHpGASl l^!WJ/pDLGUK-ScGASl 및 H. polymorpha DL1 AHpGASl ^ /pDLGUK를 얻을 수 있었다.  As a result, growth in SC-URA minimal medium: transformant H. polymorpha DL1 AHpGASl / ¾? / PDLGUK ᅳ HpGASl, H. polymorpha DL1 AHpGASl ^ 4.? / PDLGUK-HpGAS2, H. polymorpha DL1 AHpGASl ^^ Wp / pDLGUK-ScGASl and H. polymorpha DL1 AHpGASl ^ / pDLGUK could be obtained.
<4-3>유전자 삽입 균주의 생리적 특성 확인 <4-3> Confirmation of physiological characteristics of the gene insertion strain
본 발명자들은 상기 실험예 <4-2>에서 도입한 유전자로 인한 생리적 특성을 고형배지상에 순차적으로 희석된 세포배양액을 집적배양하여 확인하였다.  The present inventors confirmed the physiological characteristics due to the gene introduced in Experimental Example <4-2> by integrating the culture medium diluted in a serial medium on a solid medium.
구체적으로'， 상기 실험예 <4ᅳ2>의 형질전환체의 생장을 비교하기 위하여 상기 백터들로 각각 형질전환된 한세눌라 폴리모르파 균주들을 각각 3 ml 포도당 2% 복합 배지에 200 rpm으로 37°C에서 16시간 동안 전 배양하였다. 상기 배양한 균주들을 멸균수 1 ml에 처음 0D600 값을 1로 시작하여 연속적으로 1/10씩 희석하여 포도당 2%복합 한천배지와 세포벽 합성 저해제인 3¾칼코플로어 화이트 (Calcofluor White, CFW), 1% 콩고레드 (Congo Red, CR) 및 삼투교란제인 0.01% 도데실 황산나트륨 (Sodium Dodecyl Sulfate, SDS)이 각각 포함된 포도당 2%복합 한천배지에 1 씩 집적하여 37°C에서 이를 또는 삼일 동안 배양하였다. 또한， 열 스트레스에 대한 감수성을 확인하기 위해 포도당 2% 복합 한천배지에 집적하여 42 에서 이를 동안 배양하였다. Specifically, "the Experimental Example <4 eu 2> of the transformants respectively transformed century Cronulla poly 200 rpm to know 3 ml glucose 2%, respectively, the wave strain complex media into the vector in order to compare the growth of 37 Pre-incubated for 16 hours at ° C. The cultured strains were diluted 1/10 successively with 1 ml of sterile water, starting with the first 0D 600 value of 1, 3% Calcofluor White (CFW), 1% glucose agar complex and cell wall synthesis inhibitors. % Congo Red (CR) and osmotic perturbant 0.01% sodium dodecyl sulfate (SDS), each containing 2% glucose agar medium containing one was incubated at 37 ° C or 3 days. In addition, in order to confirm the susceptibility to heat stress was integrated in glucose 2% complex agar medium and cultured for 42 years.
그 결과, HpGASl 유전자를 보완한 균주는 야생형과 비슷한 생장 정도를 보였으며 (도 9의 4)， ^( 52유전자를 보완한 균주는 야생형보다는 느리지만 감수성 배지에서 생장함을 알 수 있었다 (도 9의 5). 반면， 사카로마이세스 세레비지애의 GAS1 유전자인 ScGASl 유전자를 보완한 균주는 생장 증대가 확인되지 않았으며， pDLGUK 공백터가 도입된 균주와 같은 정도의 생장을 보였다 (도 9의 6 및 7). 한편， 형질전환하지 않은 HpGASl 파쇄균주 보다 ScGASl 발현 백터나 pDLGUK 공백터가 도입된 균주의 성장이 약간 증대된 것을 관찰할 수 있었는데 이는 비록 복합배지라도 야생형 한세눌라 폴리모르파 균주가 飄3 파쇄 균주보다 생장이 빠르다고 보고된 것과 같은 현상으로 판단되었다. As a result, the strain supplemented with the HpGASl gene showed a similar degree of growth as the wild type (4 in FIG. 9), and ^ (strain supplemented with the 52 gene was slower than the wild type, but it was found to grow in a sensitive medium (FIG. 9). In contrast, Saccharomyces cerevisiae Strains supplemented with the GAS1 gene, ScGASl gene, did not show growth growth, and showed the same growth as the strain into which the pDLGUK void was introduced (FIGS. 6 and 7 of FIG. 9). On the other hand, it was observed that the growth of scGASl-expressing vector or pDLGUK vaccinated strain was slightly increased than that of untransformed HpGASl crushed strain. It was judged as the same phenomenon reported to grow fast.
<실험예 5> 사카로마이세스 세레비지애 (5accAari¾7yi s cerew's/ae)에서의 ScGASl 돌연변이 기능보완 확인 <Experiment 5> saccharide to make complementary ScGASl function mutations in my process three Levy jiae (5accAari¾7yi s cerew 's / ae )
<5-1> ScGASl유전자 파쇄 균주의 생장 확인  <5-1> Growth confirmation of ScGASl gene disruption strains
본 발명자들은 GAS1 유전자 파쇄에 의한 세포벽 약화에 의한 생장 저해를 사카로마이세스 세레비지애에서도 확인하였다.  The present inventors also confirmed growth inhibition by cell wall attenuation by GAS1 gene disruption in Saccharomyces cerevisiae.
구체적으로， 사카로마이세스 세레비지애 균주 C . cerevisiae BY4741)와 Specifically, Saccharomyces cerevisiae strain C. cerevisiae BY4741) and
ScGASl파쇄 균주 (5. cerevisiae BY4741 Λ 5£)를 3 ml 포도당 2%복합 배지에 200 rpm으로 30°C에서 16시간 동안 전 배양하였다.전 배양한 균주를 멸균수 1ml에 OD600 값 1에서 연속적으로 1/10씩 희석한 뒤， 포도당 2% 복합 한천배지와 0.005% 콩고레드 (Congo Red, CR)및 0.01%도데실 황산나트륨 (Sodium Dodecyl Sulfate, SDS)이 각각 포함된 포도당 2% 복합 한천배지에 1 ^씩 집적하여 30°C에서 삼일 또는 사일 등안 배양하였다. ScGASl crushed strain (5. cerevisiae BY4741 Λ 5 £) was pre-incubated in 3 ml glucose 2% complex medium at 200 rpm for 16 hours at 30 ° C. The pre-incubated strains were serially maintained at OD 600 value 1 in 1 ml of sterile water. 1/10 dilutions, then 1% glucose 2% agar medium, 0.005% Congo Red (CR) and 0.01% sodium dodecyl sulfate (SDS) ^ Were accumulated and incubated for 3 days or 4 days at 30 ° C.
<5-2> HpGASl및 유전자 삽입 균주꾀 생리적 특성 확인 <5-2> Confirmation of HpGASl and Gene Insert Strain Physiological Characteristics
본 발명자들은 사카로마이세스 세레비지애 균주를 이용하여 HpGASl 및 HpGAS2 유전자의 기능을 확인하기 위하여 상기 ᅳ유전자가 각각 클로닝된 백터 (YEp352ScGAPDHpt -HpGASl, YEp352ScGAPDHpt-HpGAS2)를 제작하여 ScGASl 파쇄 균주 (5. cerevisiae BY4741 AScGASD^] 각각 형질전환하였다.  The present inventors produced vectors (YEp352ScGAPDHpt -HpGASl, YEp352ScGAPDHpt-HpGAS2), each of which was cloned into the above genes in order to confirm the function of the HpGASl and HpGAS2 genes using Saccharomyces cerevisiae strains. cerevisiae BY4741 AScGASD ^], respectively.
구체적으로， 한세눌라 플리모르파 DL1 균주로부터 분리한 염색체 DNA를 주형으로 각각 서열번호 17및 18，서열번호 19및 20의 프라이머를 이용하여 증폭한Specifically, chromosomal DNA isolated from Hanshenula plymorpha DL1 strain Amplified using primers SEQ ID NO: 17 and 18, SEQ ID NO: 19 and 20, respectively, as a template
HpGASl, HpGAS2유전자 카세트를 제한효소 Xba/으로 처리하고， 제한효소 XZ /을 처리한 YEp352ScGAPDHpt백터에 클로닝 하였다 (도 7의 2). YEp352ScGAPDHpt백터는 2 micron origin, ^유전자와 GAPDH 프로모터를 가진 백터로써， 형질전환체는 URA3 유전자가 삽입되기 때문에 SC-URA 최소배지에서 형질전환체를 얻을 수 있다. 상기 HpGAS]7\ 삽입된 백터 (YEp352ScGAPDHpt-HpGASl), HpGAS27\ 삽입된 백터 (YEp352ScGAPDHpt-HpGAS2) 및 YEp352ScGAPDHpt 공백터를 ScGASl 파쇄 사카로마이세스 세레비지애 균주 (5. cerevisiae BY4741 AScGASD^] 각각 형질전환하였고 SC-URA 최소 배지에서 형질전환체를 얻어내어 희석한 세포배양액을 감수성 고형배지에 집적하여 생장을 비교하였다. HpGASl and HpGAS2 gene cassettes were treated with restriction enzyme Xba / and cloned into YEp352ScGAPDHpt vector treated with restriction enzyme XZ / (FIG. 2). The YEp352ScGAPDHpt vector is a vector with a 2 micron origin, a gene and a GAPDH promoter. The transformant can be obtained from the SC-URA medium because the URA3 gene is inserted. The HpGAS] 7 \ inserted vector (YEp352ScGAPDHpt-HpGASl), the HpGAS27 \ inserted vector (YEp352ScGAPDHpt-HpGAS2) and the YEp352ScGAPDHpt blank were scGASl crushed Saccharomyces cerevisiae strain (5. cerevisia SD BY474. Transformants were obtained from SC-URA minimal media, and diluted cell culture fluids were accumulated in sensitive solid media to compare growth.
그 결과， HpGASl 유전자를 보완한 5rf ¾ 유전자가 파쇄된 사카로마이세스 세레비지애 균주 (5. cereP 's/aeBY4741 ASc 5J/YEp352ScGAPI)Hp1; -HpGASl)는 야생형 (5. cerevisiae BY4741)과 유사한 정도로 생장이 회복되었으며 (도 15의 3), HpGAS2 유전자를 보완한 균주 (5. cerevisiae BY4741 ASc /Y¾)352ScGAPDHpt— HpGAS2)도 야행성 (5. cerew's/aeBY4741)과 유사한 정도로 생장이 회복되었다 (도 15의 4). 이를 통해 HpGAS2 유전자가 한세눌라 폴리모르파에서는 베타ᅳ 1,3-글루카노실트랜스글라이코실레이즈의 기능을 부분적으로 보완하는 것과 달리 사카로 마이세스 세레비지애에서는 기능을 완전히 보완하는 것을 알 수 있다. 한편, YEp352ScGAPDHpt 공백터가 도입된 대조군 균주도 ScGASl 파쇄 균주와 생장의 차이가 없었는데, 이를 통해 사카로마이세스 세레비지애는 한세눌라 폴리모르파와 달리 画3유전자 있는 균주와 圆 유전자 없는 균주의 생장차이가 없다는 것을 확인할 수 있었다 (도 15의 5). As a result, Saccharomyces cerevisiae strain (5. cereP ' s / aeBY4741 ASc 5J / YEp352ScGAPI) Hp1, in which the 5rf 3/4 gene complementing the HpGASl gene was disrupted; -HpGASl) was recovered to a similar extent as wild type (5. cerevisiae BY4741) (Fig. 3, 3), and the strain complementing the HpGAS2 gene (5. cerevisiae BY4741 ASc / Y¾) 352ScGAPDHpt—HpGAS2) was nocturnal (5. the growth was restored, so similar to the 's / aeBY4741) (4 in Fig. 15). This suggests that the HpGAS2 gene completely complements the function of beta-C1 1,3-glucanosyltransglycosylase in Hanshenula polymorpha, whereas it is fully complementary in Saccharomyces cerevisiae. have. On the other hand, the control strain in which the YEp352ScGAPDHpt vacancy was introduced had no difference in growth from the ScGASl crushed strain. Saccharomyces cerevisiae, unlike the Hansenula polymorpha, had a growth difference between the 画 3 gene and the 없는 gene-free strain. It was confirmed that there is no (5 in Fig. 15).
【수탁번호】 [Accession number]
기탁기관명 ： 한국생명공학연구원  Depositary Name ： Korea Research Institute of Bioscience and Biotechnology
수탁버호 ： KCTC12220BP 수탁일자 ： 20120524 Trusted server ： KCTC12220BP Trusted date ： 20120524
Figure imgf000027_0002
Figure imgf000027_0001
Figure imgf000027_0002
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000028_0001
특허절차상 미생물 기탁의 국제적 승인에
Figure imgf000028_0002
부다페스트 조약 In international approval of microbial deposit in patent procedure
Figure imgf000028_0002
Budapest Treaty
국제 서식  International format
규칙 7.1에 의한  According to rule 7.1
원기탁에 대한수탁증 대한민국， 대전광역시 305ᅳ 806 유성구 과학로 125  Receipt for Won Deposit 125, Sanghak-ro, Yuseong-gu, 305, Daejeon, Republic of Korea
한국생명공학연구원  Korea Research Institute of Bioscience and Biotechnology
Figure imgf000028_0003
Figure imgf000028_0003
ΒΡ/4 형식 (KCTC 제 17형식)  ΒΡ / 4 format (KCTC Model 17)

Claims

【청구의 범위】 [Range of request]
【청구항 1】  [Claim 1]
서 열번호 1로 기 재되는 HpGASl 유전자가 결손된 , 단백질 분비능이 향상된 한세눌라 폴리모르파 ( a e/ /a polymorpha) 변이 균주 .  Hansennura polymorpha (a e // a polymorpha) mutant strain with an improved protein secretion ability, lacking the HpGASl gene as set out in SEQ ID NO: 1.
【청구항 2】 [Claim 2]
제 1항에 있어서， 상기 변이 균주는 기탁번호 KCTC12220BP로 기탁된 것을 특징으로 하는 변이 균주 .  The variant strain according to claim 1, wherein the variant strain has been deposited with accession number KCTC12220BP.
【청구항 3】 [Claim 3]
1) URA3 유전자 파쇄 카세트를 상동재조합을 통해 효모 균주에 도입하는 단계 ; 및  1) introducing the URA3 gene disruption cassette into the yeast strain through homologous recombination; And
2) 상기 균주에 HpGASl 유전자 파쇄 카세트를 형 질전환한 후 상동재조합을 통해 염 색체상의 HpGASl 유전자를 파쇄하는 단계를 포함하는 단백질 분비능이 향상된 효모 균주의 제조 방법 .  2) Method for producing a yeast strain with improved protein secretion capacity comprising the step of transforming the HpGASl gene disruption cassette to the strain and then disrupting the HpGASl gene on the chromosome through homologous recombination.
【청구항 4] [Claim 4]
제 3항에 있어서， 상기 腿 3 유전자 파쇄 카세트는 HpURA3 유전자의 N 말단 절편， HpURA3 유전자의 C 말단 절편 및 Zeocin 저항성 유전자 (ZeoR) 카세트를 이용하여 제작한 파쇄카세트인 것을 특징으로 하는 방법 . The method of claim 3, wherein the VII3 gene disruption cassette is a disruption cassette prepared using an N-terminal fragment of the HpURA3 gene, a C-terminal fragment of the HpURA3 gene, and a Zeocin resistance gene (Zeo R ) cassette.
【청구항 5】 [Claim 5]
제 3항에 있어서， 상기 HpGASl 유전자 파쇄 카세트는 서 열번호 1로 기 재되는 염기서 열로 구성되는 ？^^ 유전자의 N 말단 절편， // AS 유전자의 C 말단 절편 및 1 acZ—HpURA3- 1 acZ ?\잭\트^ N-말단 절편， lacZ_HpURA3-lacZ 트^ C-말단 절편을 이 ―하여 계자하 i fl기쉐 E 91 ^ o？ ᄒ 반 ¾ According to claim 3, wherein the HpGASl gene disruption cassette is composed of a nucleotide sequence of SEQ ID NO: 1 N-terminal fragment of the? ^^ gene, // C-terminal fragment of the AS gene and 1 acZ—HpURA3- 1 acZ ? \ Jack \ ^^ N-terminal intercept, lacZ_HpURA3-lacZ ^ C-terminal intercept.
【청구항 6] [Claim 6]
1) 제 1항의 변이 균주에 목적 단백질을 코딩하는 유전자를 포함하는 백터를 도입시켜 재조합 효모 균주를 제조하는 단계 ; 및  1) preparing a recombinant yeast strain by introducing a vector comprising a gene encoding a protein of interest into the variant strain of claim 1; And
2) 상기 재조합 효모 균주를 배양하는 단계를 포함하는 외래 재조합 단백질 5 생산 방법 .  2) foreign recombinant protein 5 production method comprising the step of culturing the recombinant yeast strain.
【청구항 7] [Claim 7]
제 6항에 있어서， 상기 재조합 효모 균주는 한세눌라 속 iansenula sp.)인 것을 특징으로 하는 방법 .  7. The method according to claim 6, wherein the recombinant yeast strain is Hanshenula iansenula sp.
' 【청구항 8】 "[8.]
제 6항에 있어서， 생물반응기 (bioreactor)를 이용하여 상기 변이주를 대량 배양하는 단계를 추가로 포함하는 방법 . 5 【청구항 9】  The method of claim 6, further comprising mass culturing the mutant strain using a bioreactor. 5 [claim 9]
단백질 분비능을 향상시키기 위한， 서열번호 1로 기재되는 HpGASl 유전자가 결손된 한세눌라 폴리모르파 (fe/jsa^/a polymorpha) 변이 균주의 용도. 0  Use of a Hanshenula polymorpha (fe / jsa ^ / a polymorpha) mutant strain lacking the HpGASl gene as set forth in SEQ ID NO: 1 to improve protein secretion ability. 0
c; c;
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