KR101320059B1 - Recombinant vector, recombinant yeast containing the vector, and mass-producing method of glucose oxidase using the yeast - Google Patents

Abstract

The present invention relates to mass production of glucose oxidase using recombinant vectors and recombinant yeast. More specifically, the present invention provides a recombinant vector comprising a modified glucose oxidase gene, a recombinant yeast transformed with the recombinant vector, a recombinant yeast further comprising a bacterial hemoglobin gene in the recombinant yeast, and glucose using the recombinant yeast. It relates to a method for producing oxidase. The glucose oxidase prepared above may be used to prepare calcium acid, which may be useful for the prevention and treatment of bone metabolic diseases including osteoporosis or arthritis.

Description

Recombinant vector, recombinant yeast containing the vector, and mass-producing method of glucose oxidase using the yeast}

The present invention relates to mass production of glucose oxidase using recombinant vectors and recombinant yeast. More specifically, the present invention provides a recombinant vector comprising a modified glucose oxidase gene, a recombinant yeast transformed with the recombinant vector, a recombinant yeast further comprising a bacterial hemoglobin gene in the recombinant yeast, and glucose using the recombinant yeast. It relates to a method for producing oxidase. The glucose oxidase prepared above may be used to prepare calcium acid, which may be useful for the prevention and treatment of bone metabolic diseases including osteoporosis or arthritis.

Calcium is not only a simple component of bone, but also a very important mineral that regulates various hormones and metabolism that act on bone, and is an important mineral for preventing and treating osteoporosis in growing adolescents and adults. In addition, since organic calcium agents are absorbed into the body with higher solubility than inorganic calcium, the market for osteoporosis is rapidly reorganizing and expanding, especially organic calcium agents such as calcium gluconate. It is expected to grow to over $ 106 billion by 2050.

Conventionally, chemical oxidation, electrolysis, fermentation, and enzymatic methods are known as methods for preparing calcium organic acid, but dual chemical oxidation and electrolysis are currently rarely used due to low yield, causing industrial wastewater, and technical problems. In the case of producing calcium gluconate by fermentation method, raw materials must be pretreated, and the preservation of fermented strains and complicated purification process are problematic.

Therefore, there is an attempt to simplify the manufacturing process by using enzymes instead of microorganisms, and glucose oxidase can be cited as an enzyme that can be used for the preparation of organic acid calcium.

Glucose oxidase is an enzyme that catalyzes the reaction of oxidizing β-D-glucose to glucono-δ-lactone and simultaneously reducing oxygen molecules to hydrogen peroxide, the first in cell fluids of Aspergillus niger . (Biochem. Z. 199; 136-170, 1928). In addition, since glucose oxidase catalyzes the process of preparing organic acid calcium, such as calcium gluconate, from glucose, it can be used in the production of calcium organic acid.

Commercial production of glucose oxidase is mainly performed using Aspergillus or Penicillium strains, which are difficult to use industrially because the types of strains are limited. Therefore, in order to improve the productivity of glucose oxidase, it may be prepared by introducing into Aspergillus or yeast after genetic recombination.

For example, European Patent No. 0665291 provides a technique for recombining the Aspergillus putidus glucose oxidase gene with a promoter of glucoamylase into Aspergillus putidus, US Patent No. 5,266,688 discloses Aspergillus. It provides a technique for transforming the yeast glucose oxidase gene with the ADH2 / GAP promoter and the yeast alpha factor signal sequence, and then introduced into the yeast expression vector to transform the yeast, and Korean Patent No. 10-0328639 discloses Aspergillus. The present invention provides a technique for preparing a vector and transforming a yeast by connecting a GAL10 promoter and a GAL7 terminator to an expression plasmid consisting of a signal sequence of a glucose oxidase and a structural gene thereof.

However, prior arts still fall short of the production yield for the industrial use of glucose oxidase, and there is a need for the development of genetic recombination technology that can further increase the production yield of glucose oxidase.

Thus, the present inventors modified a glucose oxidase gene of Aspergillus niger, which is mainly used in conventional recombinant expression, and devised a new glucose oxidase gene sequence suitable for expression in yeast, and by linking a signal sequence to the gene New recombinant vectors and recombinant strains have been developed that can increase the efficiency of secreting glucose oxidase from yeast. Furthermore, the present inventors further introduced a bacterial hemoglobin gene into the recombinant strain, thereby completing a technique for improving the recombinant strain into an industrial strain, and establishing optimum culture conditions for use in industrial production.

One object of the present invention is to provide a recombinant vector comprising the glucose oxidase gene of SEQ ID NO: 1.

Another object of the present invention is to provide a recombinant yeast transformed with a recombinant vector comprising the glucose oxidase gene of SEQ ID NO: 1.

Another object of the present invention is to provide a recombinant yeast transformed with the recombinant vector, further comprising a bacterial hemoglobin gene.

It is another object of the present invention to provide a method for producing glucose oxidase comprising culturing the recombinant yeast.

It is another object of the present invention to provide a method for preparing calcium acid using the prepared glucose oxidase.

Another object of the present invention to provide a composition for the prevention and treatment of bone metabolic diseases comprising the prepared calcium acid.

Still another object of the present invention is to provide a method for treating bone metabolism disease comprising administering the prepared calcium acid to a subject.

As one aspect for achieving the above object, the present invention relates to a recombinant vector comprising a glucose oxidase gene of SEQ ID NO: 1.

As used herein, the term "glucose oxidase (GOx)" refers to an enzyme that catalyzes the reaction of oxidizing β-D-glucose into glucose-δ-lactone and simultaneously reducing oxygen molecules to hydrogen peroxide. Glucose oxidase, which has been used mainly for recombinant expression, is a glucose oxidase from Aspergillus niger, and has a molecular weight of 150 to 180 kDa and is a glycoprotein that is very tightly bound to two molecules of flan adenine dinucleotide (FAD). It consists of two identical subunits (each 70-80 kDa).

In the present invention, in order to improve the expression rate of glucose oxidase in yeast, Saccharomyces cerevisiae ) was designed to use a new gene sequence modified by the Aspergillus Niger glucose oxidase gene. The newly synthesized glucose oxidase gene in the present invention has a nucleotide sequence of SEQ ID NO: 1, and is composed of a mature glucose oxidase gene (mature GOx) excluding a signal sequence. The base sequence shows 76% homology with Aspergillus Niger's gene sequence (GenBank: J05242.1), CAI (Codon Adaptation Index) is 0.86, GC content is 53%.

Recombinant vectors of the invention may further comprise expression control elements such as promoters, initiation codons, termination codons, polyadenylation signals, enhancers, or signal sequences for membrane targeting or secretion.

In a preferred embodiment, the recombinant vector of the present invention is an optimal vector construction for improving the expression rate of glucose oxidase in yeast, including a glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, an alpha-mating factor (MFα) signal sequence and a GPD terminator. It may further include. SEQ ID NO: 2 for the MFα signal sequence used in the specific examples of the present invention, SEQ ID NO: 3 for the GPD promoter, and SEQ ID NO: 4 for the GPD terminator, respectively.

As a preferred embodiment, the recombinant vector of the present invention can be used as a base vector, which is inserted into the chromosomal DNA of yeast and stably maintained when transforming, for example, Takara's pAUR101 may be used, but is not limited thereto.

Preferably, the recombinant vector of the present invention may have the cleavage map of FIG. 1, but is not limited thereto.

As another aspect, the present invention relates to a recombinant yeast transformed with a recombinant vector comprising the glucose oxidase gene of SEQ ID NO: 1. Preferably, the yeast is Saccharomyces cerevisiae . In a specific example of the present invention, Saccharomyces cerevisiae ByGx was used as the parent strain. The parent strain ByGx is a strain of Accession No. ATCC 4003219 whose genotype is known as “MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0”.

In a specific embodiment of the present invention, the yeast ( Saccharomyces transformed with the recombinant vector of Figure 1 cerevisiae ) all showed an increase in glucose oxidase activity of 1.4 times or more and a maximum of 2.08 times of glucose oxidase activity compared to the parent strain (see Table 1). The strain with the highest increase in glucose oxidase activity was named Saccharomyces cerevisiae DG-5.

As another preferred embodiment, the recombinant yeast of the present invention is transformed with a recombinant vector comprising the nucleotide sequence of SEQ ID NO: 1, and relates to a recombinant yeast further comprising a bacterial hemoglobin gene.

In the present invention, the bacterial hemoglobin gene is additionally introduced into the yeast transformed with the recombinant vector comprising the glucose oxidase gene of SEQ ID NO: 1 to maximize the productivity of the glucose oxidase.

Preferably, the bacterial hemoglobin may be Vitreoscilla hemoglobin of Viriosila. The base sequence of the hemoglobin gene of Viriosila used in the specific example of the present invention is shown in SEQ ID NO: 5.

Preferably, the recombinant yeast of the present invention may be that the hemoglobin gene of Viriosila is inserted into the pmt1 gene region on the yeast chromosome.

In a specific embodiment of the present invention, a new recombinant yeast was introduced to the previously prepared Saccharomyces cerevisiae DG-5 strain, which additionally introduced the hemoglobin gene of Viriosila, and this was Saccharomyces cerevisiae. It was named DGVh. The DGVh strain exhibited about two times more glucose oxidase activity than the parental strain DG-5, which represents a remarkable yield improvement of about four times more than the ByGx strain corresponding to the parent strain of DG-5 ( See Table 2). Accordingly, the present inventors deposited the Saccharomyces cerevisiae DGVh strain on September 20, 2011 to Korean Colletion for Type Cultures (KCTC), Biotechnology Research Institute, Genetic Resource Center, 111, Science-ro, Yuseong-gu, Daejeon, Korea. KCTC12020BP was granted.

Thus, preferably, the recombinant yeast of the present invention may be a strain of Accession No. KCTC12020BP.

Also preferably, the recombinant yeast of the present invention may be one that includes the hemoglobin gene of Viriosila by further transformation of the recombinant vector comprising the hemoglobin of Viriosila.

More preferably, the recombinant vector comprising the hemoglobin of Viriosila may comprise a cassette consisting of the TEF1 promoter, the hemoglobin gene of Viriosila and the CYC1TT transcription terminator. In a specific embodiment of the present invention, the cassette consisting of the TEF1 promoter, Vitriola's hemoglobin gene and CYC1TT transcription terminator is named synTVHb (or TVHb), and the base sequence thereof is shown in SEQ ID NO: 6.

More preferably, the recombinant vector comprising the hemoglobin of Viriosila is a structure for stably inserting the hemoglobin gene of Viriosila by the homologous recombination into the pmt1 gene site in the chromosome of the yeast, and is homologous to the pmt1 gene. One region, a TEF1 promoter, a hemoglobin gene of Viriosila, a CYC1TT transcription terminator, a selection marker gene, and a second region homologous to the pmt1 gene. The selection marker gene may be preferably a URA3 gene.

As another aspect, the present invention relates to a method for producing glucose oxidase comprising culturing the recombinant yeast. The medium and culture conditions may be appropriately selected depending on the host cell. The conditions such as the temperature, the pH of the medium and the incubation time can be appropriately adjusted so as to be suitable for cell growth and mass production of the protein at the time of culturing.

Preferably, the present invention has established optimal culture conditions for the use of the recombinant yeast for industrial production. The culturing the recombinant yeast may include any one of the following culture conditions:

(a) the initial inoculation concentration is 10-20%,

(b) 20 to 40 g / l sucrose as carbon source,

(c) containing 20 to 40 g / l yeast extract as a nitrogen source;

(d) the stirring speed in the fermentor is 200 to 400 rpm,

(e) the oxygen supply during the culture is 1.0 to 1.6 vvm, or

(f) pH 6-7, and temperature 20-50 degreeC.

As another aspect, the present invention relates to a method for producing calcium organic acid using glucose oxidase produced by the above method. Preferably, the organic acid calcium is calcium gluconate. Glucose oxidase catalyzes the process of preparing organic acid calcium, such as calcium gluconate, from glucose, and thus can be used in the production of calcium organic acid.

As another aspect, the present invention relates to a composition for the prevention and treatment of bone metabolic diseases comprising the prepared calcium acid.

In addition, the present invention relates to a method for treating bone metabolic diseases comprising the step of administering the prepared calcium acid to the subject.

Preferably, the bone metabolic disease includes osteoporosis or arthritis.

The composition for preventing and treating bone metabolic disease of the present invention may further include a known agent for treating bone metabolic disease. Preferably, it may further comprise beta glucan (β-glucan). Beta glucan refers to a polymer of glucose (glucose) extracted from microorganisms, grains, mushrooms, seaweed, yeast, and the like. Beta glucan is known as a therapeutic agent for bone metabolic diseases such as fracture and inflammation and can be included in the composition for the treatment and prevention of bone metabolic diseases together with the organic acid calcium obtained in the present invention.

In addition, the composition may be formulated to further include a pharmaceutically acceptable carrier, for example, saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol as a pharmaceutically acceptable carrier. And one or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate injectable formulations, pills, capsules, or tablets such as aqueous solutions, suspensions, emulsions and the like.

Recombinant yeast comprising the recombinant vector of the present invention is significantly superior to the parent strain of the production yield of glucose oxidase, and thus can be utilized industrially by reducing the production cost of glucose oxidase. In addition, the glucose oxidase prepared in the present invention may be used to prepare calcium organic acid, and may be used to provide a therapeutic agent for various bone metabolic diseases including osteoporosis or arthritis.

Figure 1 shows the preparation of recombinant plasmid for the expression of glucose oxidase (hereafter GOx) gene.
2 shows S. cerevisiae BY4741, S. cerevisiae ByGx and S. cerevisiae The result of confirming GOx production amount of DG-5 by SDS-PAGE is shown.
Figure 3 shows the manufacturing process of the VHb expression cassette for introducing the hemoglobin gene (VHb) of Viriosila to the pmt1 gene on the yeast chromosome.
4 (a) shows the results of electrophoresis confirming that DNA fragments of TVHb, Pmt1U, and URA were each amplified by PCR, (b) shows the results of electrophoresis confirming that DNA fragments of TVHb-URA were amplified by PCR, (c) shows the results of electrophoresis confirming that the DNA fragment of Pmt1U-TVHb-URA was amplified by PCR, and (d) shows the results of electrophoresis confirming the presence of Pmt1U-TVHb-URA in the transformed yeast strain.
5 shows S. cerevisiae BY4741, S. cerevisiae ByGx, S. cerevisiae DG-5 and S. cerevisiae The result of comparing the molecular weight of GOx produced by DGVh is shown.
6 shows enzyme activity test results using ring size.
7 shows GOx activity with pH and temperature.
8 shows the growth and GOx activity of the cells according to the initial bacterial inoculation concentration.
9 shows the growth and GOx activity of the cells according to the type of carbon source in the medium.
10 shows the growth and GOx activity of the cells according to the concentration of the nitrogen source in the medium.
11 shows the growth and GOx activity of the cells according to the stirring speed.
12 shows the growth and GOx activity of the cells according to the amount of aeration.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Chromosome insertion Glucose  Preparation of Oxidase Gene Expression Recombinant Strains

1-1. Glucose  Oxidase Gene Synthesis

In order to increase the expression rate of glucose oxidase (hereinafter referred to as GOx) in yeast, the present inventors have artificially modified the nucleotide sequence of the GOx gene to codon usage of Saccharomyces cerevisiae , an expression host. Synthesized. The newly synthesized GOx gene in the present invention is composed of mature GOx (mature GOx) moieties except for a signal sequence, and the nucleotide sequence thereof is shown in SEQ ID NO: 1. The base sequence of the synthesized GOx gene is Aspergillus Niger niger ) showed 76% homology with GenBank (GenBank: J05242.1), CAI (Codon Adaptation Index) was 0.86, GC content was 53%.

The primer set used for amplification of the mature GOx gene is as follows.

mGOX-F: 5 '-TTG CCA CAC TAC ATC AGA TCT AAC G (SEQ ID NO: 7)

mGOX-R: 5'- ATT CAC GCT AGC TCA TCT CCG CCT GCG ACG CGA TCC TCT TTG CAT AGA AGC GTA GTC TTC CAA G (SEQ ID NO: 8)

PCR was performed using Agilent's PfuUltra II Fusion HS DNA polymerase, and PCR solution was mixed with 5 ~ 30ng template DNA, 0.2 uM primer DNA, 1mM dNTP mixture, and 1uL DNA polymerase / 50uL. Denaturation) was adjusted to 95 ℃, 72 ℃ for DNA extension (extension), the annealing (annealing) temperature was adjusted to Tm-2 ~ 3 ℃ in consideration of the Tm of each PCR primer. Synthesis time (extension time) was adjusted to the conditions of each PCR based on 15 ~ 30 seconds / kb DNA. The amplified DNA was loaded onto 0.8% (w / v) agarose gel and confirmed by electrophoresis.

1-2. Secretion signal Of peptide  Gene synthesis

In order to promote the extracellular secretion of GOx produced from the artificially synthesized GOx gene, a signal peptide of MFα (alpha-mating factor), which is known as a representative secretion signal peptide of yeast, was used. The base sequence of the gene of MFα is shown in SEQ ID NO: 2.

Artificially synthesized MFα was amplified by PCR using artificially synthesized forward primers including ribosome binding sites, etc. (51bp) upstream of the GOx gene, and reverse primers containing the N-terminal portion (26bp) of mature GOx. Afterwards, the mature GOx gene amplified in Example 1 was fused using overlapped PCR.

The primer set used for amplification of the MFα signal peptide gene is as follows.

UpGMA-F: 5'- ACT AGT GAT CAG CAA CCA GCC TTT CCT CTC TCA TTC CCT CAT CTG CCC ATC ATG AGA TTT CCT TCA ATT TTT ACT GCA (SEQ ID NO: 9)

mGMA-R: 5'- CCG TTA GAT CTG ATG TAG TGT GGC AAA TGC CAA GCT TCA GCC TCT CTT TTA (SEQ ID NO: 10)

1-3. Synthesis of Promoter Genes

As a promoter for expressing the artificially synthesized GOx gene, a GPD (glyceraldehyde-3-phosphate dehydrogenase) promoter was selected to induce representative constitutive expression of yeast. The synthesis of the promoter was obtained by amplification from genomic DNA of yeast by PCR. The base sequence of the GPD promoter is shown in SEQ ID NO: 3.

The primer set used at this time is as follows. In particular, the GPD-R primers include ribosome binding sites, which are upstream of the GOx gene, and can be easily fused with MFα and mature GOx fusions through overlapped PCR.

GPD-F: 5'-ACG TAC GAG CTC TCG AGT TTA TCA TTA TCA ATA CTC GC (SEQ ID NO: 11)

GPD-R: 5'-GAG AGG AAA GGC TGG TTG CTG ATC ACT AGT GCA CTG TCG AAA CTA AGT TCT TGG TGT TTT AAA (SEQ ID NO: 12)

1-4. Synthesis of Terminator Gene

A glyceraldehyde-3-phosphate dehydrogenase (GPD) terminator was selected as the transcription termination DNA for terminating mRNA synthesis in the expression of the artificially synthesized GOx gene. The terminator was synthesized by amplification from genomic DNA of yeast by PCR. The base sequence of the GPD terminator is shown in SEQ ID NO: 4.

The primer set used at this time is as follows. In particular, the GPDT-F primer contains the C-terminal portion of the GOx gene and can be easily fused with MFα and mature GOx fusions via overlapped PCR.

GPDT-F: 5'- GGA TCG CGT CGC AGG CGG AGA TGA GCT AGC GTG AAT TTA CTT TAA ATC TTG CAT TTA A (SEQ ID NO: 13)

GPDT-R: 5'- CAC TAC GCA TGC GCC AAA GAT TAC TCC TGT AGA ATT TG (SEQ ID NO: 14)

1-5. Preparation of Recombinant Vectors and Preparation of Recombinant Yeast

As a vector for introducing the artificially synthesized GOx gene into yeast, Takara's pAUR101, which is known to be inserted into the chromosomal DNA of the yeast and maintained stably, was used. Since the pAUR101 vector uses resistance to the antibiotic Aureobasidin A as a selection marker, it is possible to easily select a transformant in a complex medium.

The DNA fragments prepared in Examples 1-1 to 1-4 were fused in the order of GPD promoter-MFα signal peptide-mature GOx-GPD terminator using overlapped PCR to make one DNA construct, and then the pAUR101 vector. It was inserted between the SmaI and SphI site. The overall preparation of the recombinant plasmid for GOx gene expression is shown in FIG. 1.

The recombinant plasmid thus prepared was named pAURGOX, transformed into E. coli JM109, amplified in large quantities, introduced into yeast ( S. cerevisiae ByGx, ATCC 4003219) by chemical transformation, and inserted into chromosomal DNA of yeast. The recombinant yeast containing the GOx gene was constructed.

Example  2. Of Recombinant Yeast GOx  Active measurement

2-1. GOx  Active measurement

Since oxidation of GOx produces an equivalent amount of H ₂ O ₂ per 1 mole glucose, the concentration change of H ₂ O ₂ is used to measure the GOx activity of the recombinant yeast prepared in Example 1 above. It was. To a sterile medium (1M KPO ₄ , 9.5 mM glucose, agarose 0.7%), agar was added with 0.2 mM o-danisidine, a chromogen, and 10 u / ml HRP (Horse Radish Peroxidase; 250 U / mg). Plates were prepared and then inoculated with recombinant yeast. After incubating the yeast for one day at 30 ° C., 10 colonies in which brown halo around the colonies were vividly and broadly selected on the agar medium of the Petri dish plate were selected. Selected colonies were inoculated in YPD liquid medium and shaken for 2 days at 250 rpm at 30 degrees. Each culture was centrifuged at 5000 rpm for 10 minutes to take a supernatant, and then the GOx activity of each supernatant was measured and compared with the GOx activity of the parent strain ( S. cerevisiae ByGx).

Table 1 summarizes the results. As shown in Table 1, all strains showed a remarkable increase in enzyme activity of at least 1.4 times that of the parent strain ByGx. In particular, the strain named DG-5 showed the highest GOx activity, which is more than twice the result of the parent strain. In addition, the DG-5 strain showed a 1.6-fold increase in cell weight compared to the parent strain, which was determined to have increased GOx production due to higher cell growth than the parent strain.

Strain Cell weight (OD600) GOx active, U / ml Growth rate (times) Mother strain (ByGx) 9.73 10.13 ± 0.15 1.00 DG-1 16.16 17.70 ± 0.25 1.75 DG-3 14.18 11.70 ± 0.17 1.56 DG -5 16.14 21.08 ± 0.21 2.08 DG-6 14.18 17.70 ± 0.23 1.75 DG-9 16.32 15.08 ± 0.18 1.49 DG-10 12.03 14.17 ± 0.11 1.40 DG-12 16.74 16.20 ± 0.15 1.60 DG-13 14.75 16.95 ± 0.17 1.67 DG-14 16.86 16.96 ± 0.25 1.68 DG-15 14.18 17.62 ± 0.19 1.74 DG-18 14.27 15.37 ± 0.23 1.52 DG-22 11.30 14.32 ± 0.09 1.42 DG-23 13.79 16.35 ± 0.15 1.61 DG-25 17.50 15.60 ± 0.18 1.54

2-2. Of GOx  Check production

In order to confirm the production of GOx in the DG-5 strain showing the highest enzymatic activity in Example 2-1, S. cerevisiae BY4741, a cloning host, S. cerevisiae ByGx, and DG-5, a recombinant strain of GOx producing The culture supernatant was concentrated 10-fold, and each 5ul was loaded on Bio-Rad's Any kD PAGE gel and electrophoresed to compare the amount of GOx protein contained in the supernatant.

As a result, as shown in Figure 2, it was found that the intensity and clarity of the protein band of DG-5 is high, which indicates that DG-5 produces a greater amount of GOx protein than the parent strain.

Example  3. Through the introduction of bacterial hemoglobin GOx  Production of productivity enhancing strains

3-1. VHb ( Vitreoscilla hemoglobin ) Preparation of Expression Cassette

In order to further improve the GOx productivity of the recombinant yeast strain into which the GOx gene prepared in Example 1 was introduced, bacterial hemoglobin was further introduced.

The amino acid sequence of VHb (GenBank: AY278220.1) was obtained and the DNA sequence optimized for S. cerevisiae was obtained using JAVA Codon Adaptation Tool (http://www.jcat.de/), and this sequence number was 5 is shown. In addition, this was combined with the nucleotide sequence of the S. cerevisiae TEF1 promoter (GenBank: EF210199) and the CYC1TT transcription sequence of the pYES2.1 vector to complete the entire nucleotide sequence of the VHb expression cassette. The completed sequence is designed to regulate the expression of the VHb gene by the 434 bp TEF1 promoter and the 273 bp CYC1TT terminator. The length of the artificially synthesized DNA is 1148 bp, and is shown in SEQ ID NO: 6.

The completed nucleotide sequence was commissioned by Bioneer to make a DNA fragment, which was named synTVHb (or TVHb).

3-2. Yeast chromosome pmt1  For insertion into a gene region VHb  Preparation of Expression Cassettes

In order to insert the VHb expression cassette prepared in Example 3-1 into the pmt1 gene region on the yeast chromosome, a portion of the Pmt1 gene and the URA3 gene as a selection marker were prepared using PCR, and each fragment was subjected to overlapped PCR. To the two ends of the VHb expression cassette (FIG. 3).

Since the recombinant strain S. cerevisiae DG5 prepared in Example 1 is a URA3 deletion mutant strain, when the URA3 gene is inserted, the recombinant strain S. cerevisiae DG5 survives in uracil-deficient synthetic medium, thereby successfully achieving cell growth. Therefore, the strain into which the VHb gene is inserted can be easily obtained.

One) Pmt1U DNA  Synthesis of fragments

Chromosome DNA (genomic DNA) of S. cerevisiae DG5 was isolated using Zymo Research's Bacterial / Fungal Genomic DNA kit and used as template DNA. In addition, primers Pmt-UF (ATG TCG GAA GAG AAA ACG TAC AAA CG, SEQ ID NO: 15) and Pmt-UR (GGT AGA GAG ATG ACG CAA GCT GAT A, SEQ ID NO: 16) were prepared for Pmt1U DNA amplification. PCR for DNA amplification was performed using Bio-Rad's iProof HF DNA polymerase.

PCR composition with 1.0 uL of template DNA (Genomic DNA, x10 diluted), 1.0 uL of primer Pmt-UF (0.5 uM) and Pmt-UR (0.5 uM), 0.4 uL of dNTP mix (200 uM each), 5 x iProof HF Buffer 4.0 uL, 12.4 uL Sterile HPLCwater and 0.2 uL of iProof HF DNA polymerase (0.02 U / uL final) were used.

PCR was performed at 98 ° C. for 5 minutes, 30 cycles at 98 ° C., 30 seconds at 60 ° C., and 30 cycles at 72 ° C. for 20 seconds, followed by 5 minutes at 72 ° C.

The prepared 1020 bp Pmt1U DNA fragment was confirmed by electrophoresis on agarose gel. DNA identified on the agarose gel was separated and recovered using a gel extraction kit, and the concentration of each DNA was again separated by electrophoresis on the agarose gel and determined by comparing with 1kb DNA ladder of NEB (Fig. 4a).

2) TVHb DNA  synthesis

In order to connect a portion of the Pmt1 gene to the synTVHb cassette prepared in Example 3-1, the synTVHb cassette DNA prepared in Example 3-1 was used as template DNA. In addition, primers VHb-F (CGG CAT TGG CTC CAT TAT CAG CTT GCG TCA TCT CTC TAC CAC ACC TGT TGT AAT CGA GCT CAT AG, SEQ ID NO: 17) and VHb-R (CCC TTT TTT CTA CTT TTA) for amplification of the DNA AAA TGT TAG CTC CCT CCT CTT CGG CCG CAG CTT GCA AAT TAA AGCC (SEQ ID NO: 18) was prepared and used. PCR for DNA amplification was performed using Bio-Rad's iProof HF DNA polymerase, PCR conditions are as follows.

PCR composition contains 1.0 uL of template DNA (SynTVHb DNA, x100 diluted), 1.0 uL of primer VHb-F (0.5 uM) and VHb-R (0.5 uM), 0.4 uL of dNTP mix (200 uM each), 5x iProof HF Buffer 4.0 uL, 12.4 uL Sterile HPLCwater and 0.2 uL of iProof HF DNA polymerase (0.02 U / uL final) were used.

PCR was performed at 98 ° C. for 5 minutes, 30 cycles at 98 ° C., 30 seconds at 60 ° C., and 30 cycles at 72 ° C. for 20 seconds, followed by 5 minutes at 72 ° C.

The prepared 1228bp synTVHb DNA fragment was confirmed by electrophoresis on agarose gel. The DNA identified on the agarose gel was separated and recovered using a gel extraction kit, and the concentration of each DNA was again separated by electrophoresis on the agarose gel, and then compared with the 1kb DNA ladder of NEB. (FIG. 4A).

3) URA3 DNA  synthesis

To connect a portion of the Pmt1 gene to the URA3 gene, p426GPD, a S. cerevisiae expression vector purchased from ATCC, was used as template DNA. Also for amplification of the DNA primer URA-F (GAA GAG GAG GGA GCT AAC ATT TTA AAA GTA GAA AAA AGG GGG GTA ATA ACT GAT ATA ATT AAA TTG AA, SEQ ID NO: 19) and URA-R (CAA CAT TTT TTT GAC) AAC TTC TGG ATC TAG TTC ATT ACC TTC TGT GTC GAT TCG GTA ATC TCC GAA CAG AAG G (SEQ ID NO: 20) was prepared and used. PCR for DNA amplification was performed using Bio-Rad's iProof HF DNA polymerase, PCR conditions are as follows.

PCR composition 1.0 uL template DNA (p426GPD DNA, x100 diluted), 1.0 uL each of primer URA-F (0.5 uM) and URA-R (0.5 uM), 0.4 uL of dNTP mix (200 uM each), 5 x iProof HF buffer 4.0 uL, 12.4 uL Sterile HPLCwater and 0.2 uL of iProof HF DNA polymerase (0.02U / uL final) were used.

PCR was performed at 98 ° C. for 5 minutes, 30 cycles at 98 ° C., 30 seconds at 60 ° C., and 30 cycles at 72 ° C. for 20 seconds, followed by 5 minutes at 72 ° C.

The prepared 1126 bp URA DNA fragment was confirmed by electrophoresis on agarose gel. The DNA identified on the agarose gel was separated and recovered using a gel extraction kit, and the concentration of each DNA was again separated by electrophoresis on the agarose gel, and then compared with the 1kb DNA ladder of NEB. (FIG. 4A).

4) TVHb - URA DNA  Concatenation of fragments

TVHb and URA DNA fragments obtained from the PCR were mixed to a 1: 1 molar ratio and diluted 100-fold with sterile HPLC water, and the two DNA fragments were used as template DNA for overlapped PCR for binding. At this time, the amount of DNA used was about 1-5 ng, and TVHb-URA DNA fragments were linked by performing PCR divided into two steps as follows.

In the first step of PCR, PCR reaction was performed by mixing TVHb and URA DNA mixed in a 1: 1 molar ratio with the PCR composition without adding a primer. PCR Composition 1.0 μL of template DNA (TVHb & URA DNAs, x100 diluted), 0.2 uL of dNTP mix (200 uM each), 4.0 uL of 5x iProof HF buffer, 14.6 uL of Sterile HPLCwater, and iProof HF DNA polymerase (0.02U / uL final) 0.2 uL was used. PCR was performed at 98 ° C. for 5 minutes, 15 cycles were performed at 98 ° C. for 30 seconds, 50 ° C. for 30 seconds, and 72 ° C. for 20 seconds, followed by 5 minutes at 72 ° C.

Next, in step 2 of PCR, the PCR composition of step 1 was further mixed with the following PCR composition so that the final volume was 40 uL. PCR composition with 1.0 uL of primer VHb-F (0.5 uM) and URA-R (0.5 uM) each, 0.2 uL of dNTP mix (200 uM each), 4.0 uL of 5x iProof HF buffer, 13.6 uL of Sterile HPLCwater, and iProof HF DNA polymerase (0.02U / uL final) 0.2 uL was used. PCR was performed at 98 ° C. for 5 minutes, 30 cycles at 98 ° C., 45 seconds at 55 ° C., and 30 cycles at 72 ° C. for 20 seconds, followed by 5 minutes at 72 ° C.

Through the two-step PCR, amplification and preparation of 2314 bp long DNA conjugated with TVHb-URA DNA fragments were performed (FIG. 4b). The amplified DNA was electrophoresed and separated from agarose gel, followed by Zymo Research's Gel. 2314 bp long DNA was purified using Extraction Kit. The purified DNA was used as template DNA of overlapped PCR for binding to Pmt1U DNA.

5) Pmt1U - TVHb - URA DNA  Concatenation of fragments

The TVHb-URA DNA fragment obtained from the PCR and the Pmt1U DNA fragment were mixed to a 1: 1 molar ratio, and then diluted 100-fold with sterile HPLC water, and the two DNA fragments were used as template DNA for overlapped PCR for binding. It was. At this time, the amount of DNA used was about 1-5 ng, and the Pmt1U-TVHb-URA DNA fragments were linked by performing PCR divided into two steps as follows.

In the first step, PCR reaction was performed by mixing the TVHb-URA DNA fragment and Pmt1U DNA fragment mixed at a 1: 1 molar ratio with the PCR composition without adding a primer. PCR Composition PCR Composition Template DNA (Pmt1U + TVHb-URA, x100 diluted) 1.0 uL, dNTP mix (200 uM each) 0.2 uL, 5x iProof HF Buffer 4.0 uL, Sterile HPLCwater 14.6 uL and iProof HF DNA Polymerase (0.02 U / uL final) 0.2 uL was used. PCR was performed at 98 ° C. for 5 minutes, 15 cycles were performed at 98 ° C. for 30 seconds, 50 ° C. for 30 seconds, and 72 ° C. for 20 seconds, followed by 5 minutes at 72 ° C.

Next, in step 2 of PCR, the PCR composition of step 1 was further mixed with the following PCR composition so that the final volume was 40 uL. PCR compositions with 1.0 uL of primers Pmt-UF (0.5 uM) and URA-R (0.5 uM), 0.2 uL of dNTP mix (200 uM each), 4.0 uL of 5x iProof HF buffer, 13.6 uL of Sterile HPLCwater and 13.6 uL of iProof HF DNA polymerase (0.02U / uL final) 0.2 uL was used. PCR was carried out at 98 ° C. for 5 minutes, 30 cycles at 98 ° C., 45 seconds at 55 ° C., and 30 cycles at 72 ° C. for 20 seconds, followed by 5 minutes at 72 ° C.

The two-step PCR was used to amplify and prepare 3294 bp long DNA conjugated with the Pmt1U-TVHb-URA DNA fragment (FIG. 4C). The amplified DNA was electrophoresed and separated from agarose gel and then Zymo Research. 3294 bp long DNA was purified using Gel Extraction Kit. The purified DNA was used as DNA for insertion into the Pmt1 gene region in the chromosome of S. cerevisiae DG5 strain.

DNA obtained here was isolated and purified from agarose gel and directly transformed with S. cerevisiae DG5. Pmt1U-TVHb-URA DNA fragments were identified to be amplified by PCR using the respective primer sets mentioned in Materials and Methods to confirm the presence of individual DNA fragments prior to transformation.

3-3. VHb  Expression cassette pmt1  Preparation of Recombinant Yeast Inserted into Chromosome Site

200 ng of the Pmt1U-TVHb-URA DNA fragment prepared in Example 3-2 was transformed into S. cerevisiae DG5. At this time, the transformation was performed using the Yeast Transformation Kit of Zymo Research. The transformed cells were plated on uracil-deficient Yeat Synthetic medium agar plates and incubated at 30 degrees for 4 days to induce colony production.

Since the recombinant strain S. cerevisiae DG5 prepared in Example 1 is a URA3 deletion mutant strain, when the URA3 gene is inserted, the recombinant strain S. cerevisiae DG5 survives in uracil-deficient synthetic medium, thereby successfully achieving cell growth. Therefore, the generated colonies were considered to be a case where cell growth is possible by introducing Pmt1U-TVHb-URA DNA into S. cerevisiae DG5 and homologous recombination with the pmt1 gene in the chromosome to obtain the URA3 gene.

Genomic DNA was isolated from the obtained colonies and used as template DNA to confirm that Pmt1U-TVHb-URA DNA was present in the transformed strain by PCR (FIG. 4D).

Example  4. Transgenic Strains GOX  Expression level

4-1. GOx  Activity measurement of recombinant strains (qualitative analysis)

Since oxidation of GOx produced an equivalent amount of H ₂ O ₂ per 1 mole glucose, the concentration of H ₂ O ₂ was used to measure the GOx activity of the recombinant strain. Agar plates were prepared by adding 0.2 mM o-dianisidine and HRP (250 U / mg) 10u / ml to sterile medium (1M KPO ₄ , 9.5 mM glucose, agarose 0.7%), and then inoculated with recombinant yeast. After incubating the yeast for one day at 30 ° C., colonies showing clear and wide brown halo around colonies were selected on a Petri dish plate (FIG. 6).

Selected colonies were inoculated in YPD liquid medium containing Aureobasdin A, respectively, and shaken at 30 ° C for 250 days at 250 rpm. Each culture was centrifuged at 5000 rpm for 10 minutes to take a supernatant, and then the GOx activity of each supernatant was measured. The colonies with the highest increase in GOx activity were screened and named S. cerevisiae DGVh, and deposited on September 20, 2011 at KCTC (Korean Colletion for Type Cultures). Was given accession number KCTC12020BP.

Table 2 below compares the GOx activity of S. cerevisiae DGVh strain and its parent strain ( S. cerevisiae DG5). As shown in Table 2, the GOx activity of S. cerevisiae DGVh was determined to be about 2 times higher than that of S. cerevisiae DG-5, a strain before transformation. The cell concentration was also 20% higher than other strains.

This resulted in the successful expression of the VHb gene introduced into S. cerevisiae DGVh, which facilitates the oxygen supply to the cells, which activates the overall metabolic process, promotes cell growth, and improves the biosynthesis of the foreign protein GOx. It is thought to be active. Therefore, S. cerevisiae DGVh can be usefully used for mass production of GOx through high concentration culture using aeration control and fed-batch culture.

Strain Cell weight (OD600) GOx active, U / ml S. cerevisiae BY4741 9.78 ± 0.07 Not detectable S. cerevisiae ByGx 13.24 ± 0.17 10.13 ± 0.15 S. cerevisiae DG5 16.14 ± 0.09 21.08 ± 0.21 S. cerevisiae DGVh 26.19 ± 0.13 41.18 ± 0.34

4-2. Of GOx  Molecular weight and Glycosylation ( glycosylation ) Confirm

SDS-PAGE was performed to determine the molecular weight of GOx produced from S. cerevisiae DGVh. As a result, as shown in FIG. 5, the molecular weight of GOx of S. cerevisiae DGVh was measured to be about 100 kD, indicating a 10% reduction from the molecular weight of about 110 kD, which is the molecular weight of GOx produced from the previously constructed strains.

Therefore, S. cerevisiae DGVh is estimated to have slightly reduced glycosylation due to deletion of the pmt1 gene as a result of the deletion of the original pmt1 gene because the introduced DNA fragment was inserted into the pmt1 gene region.

4-3. GOx  Activity measurement of recombinant strains (quantitative analysis)

For quantitative analysis of GOx, 0.8 ml of tertiary distilled water dissolved in 0.2 mM O-Dianisidine (molecular weight: 317.21), 10 ug of HRP (250 U / mg), and 9.5 mM D-glucose were mixed. In addition, various concentrations of GOx (0, 50, 100, 250, 500, 1000, 2000 ng / 0.1 ml) were added to maintain the color reaction for 20 minutes, and the enzyme reaction was stopped by adding 0.1 ml of 4N H ₂ SO _4. Finished. After that, a calibration curve for enzyme activity was prepared through the degree of color development at absorbance 400 nm. The results are shown in Table 3.

GOx concentration (ng / 0.1ml) Absorbance (400nm) 0 0 50 0.137 100 0.331 250 0.75 500 1.2 1000 1.88 2000 3.22

Example  5. GOx  Establishment of Optimum Conditions for Industrial Production of Recombinant Strains

5-1. Optimal initial inoculation concentration

In order to investigate the optimal strain inoculation concentration for the production of GOx, inoculation concentrations of 1%, 5%, 10%, and 15% in basal medium YPD (10 g / L Yeast Extract, 20 g / L Peptone, 20 g / L Glucose), respectively Incubated with. After 7 days of incubation at the initial pH of 5.6, 200rpm, and 30 ° C, the final enzyme activity was 4.0 U / ml when the inoculation concentration was 1%, which was 30.3 U / ml appeared in ml. It was found that the initial inoculation concentration has a significant effect on the final enzyme activity (Fig. 8). Therefore the optimal strain inoculation concentration was chosen to be 10-20%, preferably 10%.

5-2. Establish and optimize media composition

In order to confirm the optimal medium composition for mass production of GOx using S. cerevisiae DGVh producing strain, we changed the existing complex medium YPD medium to semi-defined medium composition widely used in Aerobic Baker's Yeast Fermentation, and semi-defined medium. In order to supplement and modify the strain according to the present invention, the composition of the medium was optimized according to the type and concentration for the specific nutritional components such as carbon source, nitrogen source, yeast extract, and the results are shown in Table 4.

ingredient Batch medium (per l) KH ₂ PO ₄ 1 g NaNO ₃ 3.0 g MgSO ₄ · 7H ₂ O 0.25 g Yeast extract 20 g Sucrose 30 g

5-3. Carbon source  Type and concentration optimization

The effects of cell growth and GOx content were examined by varying the type and concentration of carbon sources in semi-defined medium such as glucose, sucrose, fructose and maltose. After 7 days of incubation at the initial pH of 5.6, 200rpm, and 30 ℃, the sucrose was selected as the optimal carbon source because the addition of sucrose showed the highest cell growth and GOx activity of 26.4U / ml. 9).

Next, in order to investigate the optimal concentration of sucrose was incubated in a sucrose concentration of 10 g / l, 30 g / l, 50 g / l in semi-defined medium, respectively. Incubation at the initial pH of 5.6, 200 rpm, 30 ℃ resulted in the highest cell growth rate and enzyme activity and the final enzyme activity of 38.3 U / ml when 30 g / l sucrose was added. When 1% sucrose was added, it was observed that the final enzyme amount decreased to 31.4 U / ml. In addition, when 50 g / l sucrose was added, the final enzyme amount was 28.9 U / ml. As the concentration of carbon source increased, the enzyme production was increased due to the rapid decrease of pH and the increase of inhibitor (ethanol) during the logarithmic growth stage. It was observed to receive this inhibition. The optimum sucrose concentration was therefore determined to be 20 to 40 g / l, preferably 30 g / l.

5-4. Optimization of Nitrogen Source Concentration

In order to investigate the optimal concentration of nitrogen source based on the optimal sucrose concentration of 30 g / l, incubate at 10 g / l, 20 g / l, 30 g / l, and 40 g / l yeast extract concentration in semi-defined medium, respectively. It was. Incubation at the initial pH of 5.6, 200 rpm and 30 ℃ resulted in the highest cell growth rate and enzyme activity and the final enzyme activity of 43.6 U / ml when 20 g / l yeast extract was added. When the yeast extract was added at low concentration, the enzyme production was observed to be decreased. When 40 g / l yeast extract was added, the final enzyme amount was 42.8 U / ml. In other words, when comparing the enzyme activity of 30 g / l and 40 g / l with respect to the nitrogen source concentration of 20 g / l, the optimal yeast extract concentration was 20-40 g / l because there was no significant difference in the final cell growth or enzyme activity. , Preferably 20 g / l (FIG. 10).

5-5. 5L- Fermentor Culture conditions through Stirring  speed)

In order to observe the effect of the stirring speed during the cultivation by varying the stirring speed at the optimum conditions of the carbon source (sucrose concentration 30 g / l) and nitrogen source (yeast extract 30 g / l) (100 rpm, 200 rpm, 300 rpm, 400 rpm) The culture was carried out under the condition of 30 ° C and 1 vvm. As a result, there was no significant difference in cell growth when the absorbance and final enzyme activity of 200rpm and 300rpm were compared, but the final enzyme activity was higher than 40.1 U / ml of 300rpm at 42.8 U / ml at 200rpm. However, at 400 rpm, the enzyme activity decreased due to high shear stress and cell demage. Therefore, the most suitable stirring speed was determined at 200 to 400 rpm, preferably 200 rpm (FIG. 11).

5-6. Of Aeration  optimization

In order to examine the effect of the aeration rate during the cultivation, the aeration rate was varied (0.5 vvm, 1.0 vvm, 1.5 vvm) at the optimum conditions of carbon source (sucrose concentration 30 g / l) and nitrogen source (yeast extract 30 g / l). ) Culture was performed. As a result, the final enzyme activity was 44.8 U / ml and 45.2 U / ml when the enzyme activity was compared at 1.0 vvm and 1.5 vvm. This is because when the cell concentration is high, oxygen consumption exceeds the feed rate, and growth is limited. Since the cell growth is almost the same at 1.0 vvm and 1.5 vvm, there is no significant difference in aeration. Therefore, the optimum oxygen supply amount was determined to be 1.0 to 1.6 vvm, preferably 1.0 vvm (FIG. 12).

Korea Biotechnology Research Institute KCTC12020BP 20110920

Attach an electronic file to a sequence list

Claims (19)

A recombinant vector comprising the glucose oxidase gene of SEQ ID NO: 1.
The recombinant vector of claim 1, further comprising a glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, an alpha-mating factor (MFa) signal sequence, and a GPD terminator.
The recombinant vector of claim 1, wherein the recombinant vector has a cleavage map of FIG. 1.
Recombinant yeast transformed with a recombinant vector comprising a glucose oxidase gene of SEQ ID NO: 1.
The recombinant yeast of claim 4, wherein the recombinant vector further comprises a GPD promoter, an alpha-mating factor (MFa) signal sequence, and a GPD terminator.
The recombinant yeast of claim 4, wherein the recombinant vector has a cleavage map of FIG. 1.
The recombinant yeast of claim 4, further comprising a bacterial hemoglobin gene.
The recombinant yeast of claim 7, wherein the bacterial hemoglobin is Vitreoscilla hemoglobin.
The recombinant yeast of claim 8, wherein the hemoglobin gene of Viriosila is inserted at the pmt1 gene site on the yeast chromosome.
8. The recombinant yeast of claim 7, further transformed with a recombinant vector comprising hemoglobin of Viriosila.
The recombinant yeast of claim 10, wherein the recombinant vector comprising hemoglobin of Viriosila comprises a cassette consisting of a TEF1 promoter, a hemoglobin gene of Viriosila, and a CYC1TT terminator.
The recombinant vector comprising the hemoglobin of Viriosila is homologous to the first region homologous to the pmt1 gene, the TEF1 promoter, the hemoglobin gene of the Viriosila, the CYC1TT terminator, the selection marker gene, and the pmt1 gene. Recombinant yeast comprising a second region.
The method of claim 7, wherein the recombinant yeast Saccharomyces ( Saccharomyces) cerevisiae ) recombinant yeast, which is KCTC12020BP.
A method for preparing glucose oxidase, comprising culturing the recombinant yeast of any one of claims 4 to 13.
The method for preparing glucose oxidase according to claim 14, wherein the recombinant yeast is cultivated under any of the following culture conditions:
(a) the initial inoculation concentration is 10-20%,
(b) 20 to 40 g / l sucrose as carbon source,
(c) containing 20 to 40 g / l yeast extract as a nitrogen source;
(d) the stirring speed in the fermentor is 200 to 400 rpm,
(e) the oxygen supply during the culture is 1.0 to 1.6 vvm, or
(f) pH 6-7, and temperature 20-50 degreeC.
A method for producing calcium organic acid using the glucose oxidase prepared by claim 14.
A composition for the prevention and treatment of bone metabolic diseases comprising calcium organic acid prepared by claim 16.
The composition of claim 17, wherein the bone metabolic disease is osteoporosis or arthritis.
18. The composition for preventing and treating bone metabolism disease according to claim 17, further comprising beta glucan.

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