WO2013058516A1

WO2013058516A1 - Recombinant vector, recombinant yeast containing the vector, and mass-producing method of glucose oxidase using the yeast

Info

Publication number: WO2013058516A1
Application number: PCT/KR2012/008431
Authority: WO
Inventors: Hyung-Rae Cho; Seung-Bae MOON; Ki-Young Kim; Joo-Wan Kim; Jong-Min Lim; Chae-Hun Ra; Hyun-Dong Shin; Sae-Kwang Ku; Jae-Hak Sohn; Dong-Gun SHIN
Original assignee: Glucan Corporation
Priority date: 2011-10-18
Filing date: 2012-10-16
Publication date: 2013-04-25
Also published as: KR20130042346A; KR101320059B1

Abstract

The present invention relates to a method for mass-producing glucose oxidase using a recombinant vector and a recombinant yeast. More particularly, the present invention relates to 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, and a method for producing glucose oxidase using the recombinant yeast. The glucose oxidase produced by the above method can be used for the preparation of organic acid calcium, which is useful in the prevention or treatment of metabolic bone diseases including osteoporosis or arthritis.

Description

RECOMBINANT VECTOR, RECOMBINANT YEAST CONTAINING THE VECTOR, AND MASS-PRODUCING METHOD OF GLUCOSE OXIDASE

USING THE YEAST

[Technical Field]

The present invention relates to a method for mass-producing glucose oxidase using a recombinant vector and a recombinant yeast. More particularly, the present invention relates to 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 a method for producing glucose oxidase using the recombinant yeast. The glucose oxidase produced by the above method can be used for the preparation of organic acid calcium, which is useful in the prevention and treatment of metabolic bone diseases including osteoporosis or arthritis.

[Background Art]

Calcium is a very important inorganic substance that constitutes the bone and regulates various hormones acting on the bone and metabolism, and is also an important mineral in the prevention and treatment of both juvenile and adult osteoporosis. Organic calcium is absorbed better by the human body than inorganic calcium, and thus organic calcium such as calcium gluconate has received much attention and rapidly expanded in the osteoporosis drug market. The global osteoporosis drug market is expected to grow more than 6.0% annually by 2015 and reach 106 billion dollars in 2050.

The known preparation methods of organic acid calcium include chemical oxidation, electrolysis, fermentation and enzymatic methods. Currently, the chemical oxidation and electrolytic process are rarely used because of low yield, industrial waste water, and technical problems. Production of calcium gluconate by fermentation is problematic in pretreatment of raw materials, storage of fermentation strains, and complicated purification process.

Therefore, there is an attempt to simplify the preparation process by using enzymes instead of microorganisms. The enzyme to be used in the preparation of organic acid calcium can be exemplified by glucose oxidase.

Glucose oxidase is an enzyme that catalyzes the oxidation of β-D-glucose to glucono-5-lactone, concomitant with the reduction of oxygen to hydrogen peroxide, and it was first found in Aspergillus niger extracts (Biochem. Z. 199; 136- 170, 1928). In addition, since glucose oxidase catalyzes the preparation process of organic acid calcium, such as calcium gluconate, from glucose, it can be used in the preparation of organic acid calcium.

Commercial production of glucose oxidase is usually conducted by using strains belonging to the genus Aspergillus or Penicillium. However, there is a limitation in the type of production strain, which creates difficulties in the industrial application. Therefore, genetic recombination is first performed, and the recombinant gene is introduced into the genus Aspergillus or yeast to enhance glucose oxidase productivity.

For example, EP0665291 discloses that recombination of the glucose oxidase gene from Aspergillus foetidus and the glucoamylase promoter is performed and the recombinant gene is introduced into Aspergillus foetidus. US Patent No. 5,266,688 discloses that the glucose oxidase gene from Aspergillus niger is linked to the ADH2/GAP promoter and the yeast alpha- factor signal sequence, and introduced into a yeast expression vector to perform yeast transformation. Korean Patent No. 10-0328639 discloses a technique of transforming yeast with a vector that is prepared by linking the GAL10 promoter and GAL7 terminator to an expression plasmid consisting of the glucose oxidase signal sequence of Aspergillus niger and its structural gene.

However, these conventional techniques still do not meet the yield required for industrial-scale production of glucose oxidase, and thus there is a need to develop a genetic recombination technique for the improvement of glucose oxidase production. [Disclosure]

[Technical Problem]

Accordingly, the present inventors designed a novel glucose oxidase gene sequence suitable for yeast expression by modifying the glucose oxidase gene from Aspergillus niger, which has been frequently used in the conventional recombinant expression systems. They linked a signal sequence to the gene so as to develop a novel recombinant vector and recombinant strain for the improvement of the extracellular secretion efficiency of glucose oxidase from yeast. Moreover, the present inventors further introduced a bacterial hemoglobin gene into the recombinant strain, thereby completing a technology of improving the recombinant strain for industrial use, and also established the optimal culture conditions for industrial-scale production.

[Technical Solution]

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

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

Still another object of the present invention is to provide a recombinant yeast that is transformed with the recombinant vector and further comprises a bacterial hemoglobin gene.

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

Still another object of the present invention is to provide a method for producing organic acid calcium using the produced glucose oxidase.

Still another object of the present invention is to provide a composition for the prevention and treatment of metabolic bone diseases, comprising the produced organic acid calcium.

Still another object of the present invention is to provide a method for treating metabolic bone diseases, comprising the step of administering the produced organic acid calcium into a subject.

[Best Mode]

In one embodiment to achieve the above objects, the present invention relates to a recombinant vector including a glucose oxidase gene of SEQ ID NO. 1.

As used herein, the term "glucose oxidase (GOx)" refers to an enzyme that catalyzes the oxidation of β-D-glucose to glucono-5-lactone, concomitant with the reduction of oxygen to hydrogen peroxide. The glucose oxidase frequently used in the conventional recombinant expression systems is a glucose oxidase from Aspergillus niger, which is a glycoprotein with a molecular weight of 15CM 80 kDa and two molecules of FAD (flavin adenine dinucleotide) tightly bound, and consists of two identical subunits (each 70-80 kDa).

In order to enhance the expression level of glucose oxidase in yeast, the present inventor designed a new base sequence by modifying the glucose oxidase gene from Aspergillus niger according to the codon usage of yeast {Saccharomyces cerevisiae). The newly synthesized glucose oxidase gene of the present invention has the base sequence of SEQ ID NO. 1, and consists of a mature glucose oxidase gene (mature GOx) excluding a signal sequence. The base sequence shows 76% homology with that of Aspergillus niger (GenBank: J05242.1) and has CAI (Codon Adaptation Index) of 0.86 and GC content of 53%.

The recombinant vector of the present invention may further include expression regulatory elements, such as a promoter, an initiation codon, a stop codon, a polyadenylation signal and an enhancer, or signal sequences for membrane targeting or secretion.

In the preferred embodiment, the recombinant vector of the present invention may further include a GPD (glyceraldehyde-3 -phosphate dehydrogenase) promoter, an MFa (alpha-mating factor) signal sequence and a GPD terminator as an optimal vector for enhancement of the expression level of glucose oxidase in yeast. The base sequences of the MFa signal sequence, the GPD promoter, and the GPD terminator used in the specific Example of the present invention are represented by SEQ ID NOs. 2, 3, and 4, respectively.

In the preferred embodiment, a vector that is able to integrate and stably maintain in the yeast chromosomal DNA upon transformation may be used as a basic vector for the recombinant vector of the present invention, and exemplified by Takara pAUR.101, but is not limited thereto.

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

In another embodiment, the present invention relates to a recombinant yeast that is transformed with the recombinant vector including the glucose oxidase gene of SEQ ID NO. 1. Preferably, the yeast is Saccharomyces cerevisiae. In the specific Example of the present invention, Saccharomyces cerevisiae ByGx was used as a parental strain. The parental strain is a strain of Accession No. ATCC 4003219, and its genotype is known as "MATa his3Al leu2A0 metl5A0 ura3A0".

In the specific Example of the present invention, all of the yeasts {Saccharomyces cerevisiae) transformed with the recombinant vector of FIG. 1 showed 1.4 times, and as much as 2.08 times, increased glucose oxidase activity, compared to the parental strain (see Table 1). The strain showing the highest glucose oxidase activity was designated as Saccharomyces cerevisiae DG-5.

In still another preferred embodiment, the present invention relates to a recombinant yeast that is transformed with the recombinant vector including the base sequence of SEQ ID NO. 1 and further includes a bacterial hemoglobin gene.

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

Preferably, the bacterial hemoglobin may be Vitreoscilla hemoglobin. A base sequence of the Vitreoscilla hemoglobin gene used in the specific Example of the present invention is represented by SEQ ID NO. 5. Preferably, the recombinant yeast of the present invention may be prepared by inserting the Vitreoscilla hemoglobin gene at the site of a pmtl gene on the yeast chromosome.

In the specific Example of the present invention, the Vitreoscilla hemoglobin gene is additionally introduced into the previously prepared Saccharomyces cerevisiae DG-5 strain to prepare a new recombinant yeast, designated as Saccharomyces cerevisiae DGVh. The glucose oxidase activity of the DGVh strain was approximately two times higher than that of the parental strain DG-5, which is surprisingly four times higher than that of the ByGx strain that is a parental strain of DG-5 (see Table 2). Therefore, the present inventors deposited the Saccharomyces cerevisiae DGVh strain at KCTC (Korean Collection for Type Cultures, Genetic Resources Center, Korea Research Institute of Bioscience and Biotechnology, 111 Gwahangno, Yuseong-gu, Daejeon) under Accession No. KCTC12020BP on Sep. 20, 2011.

Preferably, the recombinant yeast of the present invention may be the strain with Accession No. KCTC 12020BP.

Preferably, the recombinant yeast of the present invention may further include the Vitreoscilla hemoglobin gene by transformation with the recombinant vector including the Vitreoscilla hemoglobin gene.

More preferably^ the recombinant vector including the Vitreoscilla hemoglobin gene may include a cassette consisting of a TEF1 promoter, the Vitreoscilla hemoglobin gene, and a CYC ITT transcription terminator. In the specific Example of the present invention, the cassette consisting of the TEF1 promoter, the Vitreoscilla hemoglobin gene, and the CYC ITT transcription terminator was designated as synTVHb (or TVHb), and its base sequence is represented by SEQ ID NO. 6.

More preferably, the recombinant vector including the Vitreoscilla hemoglobin gene may include a first region homologous to the pmtl gene, the TEF1 promoter, the Vitreoscilla hemoglobin gene, the CYC ITT transcription terminator, a selection marker gene, and a second region homologous to the pmtl gene, in order to stably insert the Vitreoscilla hemoglobin gene at the site of the pmtl gene on the yeast chromosome by homologous recombination. Preferably, the selection marker gene may be a URA3 gene.

In still another embodiment, the present invention relates to a method for producing glucose oxidase, including the step of culturing the recombinant yeast. The appropriate medium and culture conditions may be selected according to the host cell. Upon cultivation, the conditions such as temperature, pH of the medium, and culture time can be controlled to be suitable for cell growth and mass-production of the protein.

Preferably, the present inventors established optimal culture conditions for the industrial production of the recombinant yeasts. The step of culturing the recombinant yeast may include any one of the following culture conditions:

(a) 10 to 20% of initial inoculation concentration,

(b) 20 to 40 g/1 of sucrose as a carbon source,

(c) 20 to 40 g/1 of yeast extract as a nitrogen source,

(d) 200 to 400 rpm of agitation speed in a fermentor,

(e) 1.0 to 1.6 wm of oxygen feed during cultivation, or

(f) at pH 6 to 7 and temperature of 20 to 50°C.

In still another embodiment, the present invention relates to a method for producing organic acid calcium using the glucose oxidase produced by the above method. Preferably, the organic acid calcium is calcium gluconate. Since glucose oxidase catalyzes the preparation process of organic acid calcium, such as calcium gluconate, from glucose, it can be used in the preparation of organic acid calcium.

In still another embodiment, the present invention relates to a composition for the prevention and treatment of metabolic bone diseases, including the produced organic acid calcium.

Further, the present invention relates to a method for treating metabolic bone diseases, including the step of administering the produced organic acid calcium into a subject.

Preferably, the metabolic bone diseases include osteoporosis or arthritis.

The composition for the prevention and treatment of metabolic bone diseases of the present invention may further include the known therapeutic agent for metabolic bone disease. Preferably, it may further include beta glucan (β-glucan). Beta glucan refers to a glucose polymer extracted from microorganism, grain, mushroom, seaweed, yeast or the like. Beta glucan is known as a therapeutic agent for metabolic bone diseases such as bone fracture and inflammation, and thus it may be included in the composition for the prevention and treatment of metabolic bone diseases, together with the organic acid calcium produced in the present invention.

Further, the composition of the present invention may be prepared by further including a pharmaceutically acceptable carrier. Examples of the pharmaceutically acceptable carrier include a saline solution, sterile water, a Ringer's solution, a buffered saline solution, a dextrose solution, a maltodextrin solution, glycerol, ethanol and a mixture of one or more thereof. If necessary, the composition may also include other conventional additives such as antioxidants, buffers, and bacteriostatic agents. Moreover, the composition may additionally include diluents, dispersants, surfactants, binders, and lubricants to be formulated into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules or tablets.

[Advantageous Effects]

A recombinant yeast including a recombinant vector of the present invention shows excellent production yield of glucose oxidase, compared to a parental strain, thereby reducing production cost of glucose oxidase, and thus it can be a cost effective means for industrial production. Further, the glucose oxidase produced in the present invention is used in the preparation of organic acid calcium, thereby providing a therapeutic agent for various metabolic bone diseases including osteoporosis or arthritis.

[Description of Drawings]

FIG. 1 shows a preparation process of a recombinant plasmid for glucose oxidase (hereinafter, referred to as GOx) gene expression;

FIG. 2 is the result of SDS-PAGE showing the GOx productivity of S. cerevisiae BY4741, S. cerevisiae ByGx, and S. cerevisiae DG-5;

FIG. 3 shows a preparation process of a VHb expression cassette for introduction of a Vitreoscilla hemoglobin gene at the site of a pmtl gene on the yeast chromosome;

FIG. 4(a) is the result of electrophoresis showing PCR amplification of each

DNA fragment of TVHb, PmtlU and URA, (b) is the result of electrophoresis showing PCR amplification of a DNA fragment of TVHb-URA, (c) is the result of electrophoresis showing PCR amplification of a DNA fragment of PmtlU-TVHb-URA, and (d) is the result of electrophoresis showing the presence of PmtlU- TVHb-URA in the transformed yeast strain;

FIG. 5 is the result of comparing the molecular weights of GOx produced in S. cerevisiae BY4741, S. cerevisiae ByGx, S. cerevisiae DG-5, and S. cerevisiae DGVh;

FIG. 6 is the result of enzymatic activity test based on the halo size;

FIG. 7 shows GOx activity according to pH and temperature;

FIG. 8 shows cell growth and GOx activity according to initial inoculation concentration;

FIG. 9 shows cell growth and GOx activity according to the type of carbon source in the medium;

FIG. 10 shows cell growth and GOx activity according to the concentration of nitrogen source in the medium;

FIG. 1 1 shows cell growth and GOx activity according to the agitation speed; and

FIG. 12 shows cell growth and GOx activity according to the aeration level. [Mode for Invention]

Hereinafter, the present invention will be described in detail with reference to Examples. However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples. Example 1. Preparation of Recombinant Strain Expressing Chromosomal Integrating Glucose Oxidase Gene

1-1. Synthesis of GOx gene

In order to improve the expression level of glucose oxidase (hereinafter, referred to as GOx) in yeast, the present inventors synthesized an artificial sequence by modifying the base sequence of GOx gene to be suitable for the codon usage of an expression host, Saccharomyces cerevisiaea. The newly synthesized GOx gene of the present invention consists of mature GOx excluding a signal sequence, and its base sequence is represented by SEQ ID NO. 1. The base sequence of the synthesized GOx gene showed 76% homology with that of Aspergillus niger (GenBank: J05242.1) and had CAI (Codon Adaptation Index) of 0.86 and GC content of 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 a PfuUltra II Fusion HS DNA polymerase provided by Agilent, and a PCR solution was prepared by mixing 5~30 ng of template DNA, 0.2 μΜ of each primer DNA, 1 mM-dNTP mixture and 1 μΐ, DNA polymerase/50 uL, and DNA denaturation was performed at 95°C, DNA extension at 72°C, and annealing at Tm-2~3°C considering Tm of each PCR primer. The extension time was adjusted according to each PCR condition, based on 15~30 sec/kb DNA. The amplified DNA was loaded on a 0.8% (w/v) agarose gel, and then examined by electrophoresis.

1-2. Gene Synthesis for Secretion Signal Peptide

For extracellular secretion of GOx to be produced from the artificially synthesized GOx gene, an MFa (alpha-mating factor) signal peptide known as a representative yeast secretion signal peptide was used. A base sequence of the MFa gene is represented by SEQ ID NO. 2.

The artificially synthesized MFa was amplified by PCR using an artificially synthesized forward primer including a ribosome binding site (51 bp) upstream of the GOx gene and a reverse primer including the N-terminus (26 bp) of the mature GOx, and then fused with the mature GOx gene amplified in Example 1 by overlapped PCR.

In this regard, the primer set used for amplification of the MFa 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 Gene

A GPD (glyceraldehyde-3 -phosphate dehydrogenase) promoter that allows constitutive gene expression in yeast was chosen as a promoter for expressing the artificially synthesized GOx gene. The promoter was obtained from the yeast genomic DNA by PCR amplification. A base sequence of the GPD promoter is represented by SEQ ID NO. 3.

The primer set used herein is as follows. In particular, a ribosome binding site upstream of the GOx gene is included in the GPD-R primer, and thus it can be easily fused with the MFa and mature GOx fusion by 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 GPD (glyceraldehyde-3 -phosphate dehydrogenase) terminator was chosen as a transcription terminator DNA for terminating mRNA synthesis upon expression of the artificially synthesized GOx gene. The terminator was obtained from the yeast genomic DNA by PCR amplification. A base sequence of the GPD terminator is represented by SEQ ID NO. 4.

The primer set used herein is as follows. In particular, the C-terminus of the GOx gene is included in the GPDT-F primer, and thus it can be easily fused with the MFa and mature GOx fusion by 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 Vector and Preparation of Recombinant

Yeast

Takara pAURlOl, which is known to integrate and stably maintain in the yeast chromosomal DNA upon transformation, was used as a vector for introducing the artificially synthesized GOx gene into yeast. Since the pAURlOl vector includes an aureobasidin A resistance gene as a selection marker, transformants can be easily selected on the complex media.

Each of the DNA fragments prepared in Examples 1-1 to 1-4 was fused in this order of GPD promoter-MFa signal peptide-mature GOx-GPD terminator by overlapped PCR so as to prepare a single DNA construct, and then inserted into the Smal and Sphl sites of the pAURlOl vector. The overall preparation process of the recombinant plasmid for GOx gene expression is shown in FIG. 1.

The prepared recombinant plasmid was designated as pAURGOX, and it was transformed into E. coli JM109 for amplification, and introduced into the yeast chromosomal DNA (S. cerevisiae ByGx, ATCC 4003219) by chemical transformation so as to construct a recombinant yeast including the GOx gene.

Example 2. Measurement of GOx Activity of Recombinant Yeast

2-1. GOx Activity Assay GOx catalyzes the oxidation of 1 mole of glucose to produce an equal amount (1 mole) of H₂0₂. In order to measure the GOx activity of the recombinant yeast prepared in Example 1, therefore, changes in the H₂0₂ concentration were measured. 0.2 mM o-dianisidine served as a chromogen and 10 μ/ml of HRP (Horse Radish Peroxidase; 250 U/mg) were added to a sterilized medium (1 M KP0₄, 9.5 mM glucose, agarose 0.7%) to prepare agar plates, and the recombinant yeast was inoculated thereto. After cultivating the recombinant yeast for 1 day at 30°C, 10 colonies surrounded by a clear large brown halo on the agar in Petri dishes were selected. Each of the selected colonies was inoculated in YPD liquid media, and shaking-cultured at 30°C and 250 rpm for 2 days. Each of the culture media was centrifuged at 5000 rpm for 10 minutes to collect the supernatant. GOx activity of each supernatant was measured, and compared with that of the parental strain (S. cerevisiae ByGx).

The results are summarized in the following Table 1. As shown in Table 1 , the enzymatic activity of all strains was surprisingly 1.4 times higher than that of the parental strain ByGx. In particular, the strain designated as DG-5 showed the highest GOx activity, which was two times higher than that of the parental strain. In addition, the DG-5 strain showed approximately 1.6 times higher cell density than the parental strain. The result suggests that the increased GOx production is attributed to the higher growth rate of the mutant strain than that of the parental strain.

[Table 1 ]

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. Examination of GOx Production Amount

In order to examine GOx production in the DG-5 strain showing the highest enzymatic activity in Example 2-1, culture supernatants of the cloning host, S. cerevisiae BY4741 and the GOx-producing recombinant strain, S. cerevisiae ByGx and DG-5 were concentrated to 10-fold, and each 5 ul was loaded on an Any kD PAGE gel provided by Bio-Rad and electrophoresis was performed to compare the GOx protein amounts contained in the supernatants.

As shown in FIG. 2, the protein band of DG-5 showed higher size and intensity, indicating that DG-5 has a higher GOx productivity than the parental strain.

Example 3.Preparation of GOx Productivity-Improved Strain by Introduction of Bacterial Hemoglobin

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

In order to more improve the GOx productivity of the GOx gene-introduced recombinant yeast strain prepared in Example 1, a bacterial hemoglobin gene is intended to be introduced.

The VHb amino acid sequence (GenBank: AY278220.1) was obtained, and a DNA base sequence optimized to S. cerevisiae was obtained using a JAVA Codon Adaptation Tool (http://www.jcat.de/), which is represented by SEQ ID NO. 5. This base sequence was linked with the base sequence of S. cerevisiae TEFl promoter (GenBank: EF210199) and the CYC ITT transcription terminator of the pYES2.1 vector so as to complete the entire base sequence of VHb expression cassette. The base sequence was designed such that the VHb expression was controlled by the TEFl promoter of 434 bp and the CYC ITT terminator of 273 bp. The artificially synthesized DNA has a length of 1148 bp, and it is represented by SEQ ID NO. 6.

A DNA fragment of the completed base sequence was prepared by BIONEER Co., and designated as synTVHb (or TVHb).

3-2. Preparation of VHb Expression Cassette for Insertion at Site of pmtl gene on Yeast Chromosome

In order to insert the VHb expression cassette prepared in Example 3-1 at the site of a pmtl gene on the yeast chromosome, a part of the Pmtl gene and a URA3 gene as a selection marker were prepared by PCR, and each fragment was ligated at both ends of the VHb expression cassette by overlapped PCR (FIG. 3).

The recombinant strain, S. cerevisiae DG5 prepared in Example 1 is a URA3 deletion mutant strain. Thus, when the URA3 gene is inserted thereto, it can grow successfully in a synthetic medium lacking uracil, thereby easily obtaining the VHb gene-inserted strain.

1) Synthesis of PmtlU DNA Fragment

The genomic DNA of S. cerevisiae DG5 was isolated using a Bacterial/Fungal Genomic DNA kit provided by Zymo Research Corp., and used as a template DNA. For amplification of PmtlU DNA, a primer set of 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) was prepared and used. PCR for DNA amplification was performed using iProof HF DNA polymerase provided by Bio-Rad. 1.0 μΐ. of a template DNA (Genomic DNA, xlO diluted), each 1.0 μΐ, of the primers, Pmt-UF (0.5 μΜ) and Pmt-UR (0.5 μΜ), 0.4 μΐ of dNTP mix (200 μΜ each), 4.0 μί of 5x iProof HF buffer, 12.4 μΐ, of sterile HPLC water and 0.2 μΐ. of iProof HF DNA polymerase (0.02 ΙμΙ_^ final) were used as a PCR composition.

PCR was performed at 98°C for 5 minutes, followed by 30 cycles of at 98°C for

30 seconds, at 60°C for 30 seconds, and at 72°C for 20 seconds, and then at 72°C for 5 minutes.

The prepared PmtlU DNA fragment of 1020 bp was examined by electrophoresis on an agarose gel. The DNA on the agarose gel was isolated and recovered using a gel extraction kit, and the DNA concentration was examined by . electrophoresis on an agarose gel, and compared with 1 kb DNA ladder (NEB) (FIG. 4a).

2) Synthesis of TVHb DNA

In order to link a part of the Pmtl gene to the synTVHb cassette prepared in

Example 3-1, the synTVHb cassette DNA prepared in Example 3-1 was used as a template DNA. For DNA amplification, a primer set of 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 AAA TGT TAG CTC CCT CCT CTT CGG CCG CAG CTT GCA AAT TAA AGCC, SEQ ID NO. 18) was constructed and used. PCR for DNA amplification was performed using iProof HF DNA polymerase provided by Bio-Rad, and PCR conditions are as follows.

1.0 μΐ. of a template DNA (SynTVHb DNA, xlOO diluted), each 1.0 μΐ. of the primers, VHb-F (0.5 μΜ) and VHb-R (0.5 μΜ), 0.4 μΐ. of dNTP mix (200 μΜ each), 4.0 μΐ, of 5x iProof HF buffer, 12.4 μΐ, of sterile HPLC water and 0.2 μΐ of iProof HF DNA polymerase (0.02 ΜΙμΙ, final) were used as a PCR composition.

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

The prepared synTVHb DNA fragment of 1228 bp was examined by electrophoresis on an agarose gel. The DNA on the agarose gel was isolated and recovered using a gel extraction kit, and the DNA concentration was examined by electrophoresis on an agarose gel, and compared with 1 kb DNA ladder (NEB) (FIG. 4a).

3) Synthesis of URA3 DNA

In order to link a part of the Pmtl gene to the URA3 gene, a S. cerevisiae expression vector p426GPD purchased from ATCC was used as a template DNA. For DNA amplification, a primer set of 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 constructed and used. PCR for DNA amplification was performed using iProof HF DNA polymerase provided by Bio-Rad, and PCR conditions are as follows.

1.0 μΐ. of a template DNA (p426GPD DNA, xlOO diluted), each 1.0 uL of the primers, URA-F (0.5 μΜ) and URA-R (0.5 μΜ), 0.4 μΐ, of dNTP mix (200 μΜ each), 4.0 μΐ of 5x iProof HF buffer, 12.4 LL of sterile HPLC water and 0.2 \iL of iProof HF DNA polymerase (0.02 U/μΙ, final) were used as a PCR composition.

The prepared URA DNA fragment of 1126 bp was examined by electrophoresis on an agarose gel. The DNA on the agarose gel was isolated and recovered using a gel extraction kit, and the DNA concentration was examined by electrophoresis on an agarose gel, and compared with 1 kb DNA ladder (NEB) (FIG. 4a). 4) Ligation of TVHb-URA DNA Fragment

The TVHb and URA DNA fragments prepared by PCR were mixed with each other at a molar ratio of 1 :1, and then diluted 100-fold with sterile HPLC water to be used as a template DNA in overlapped PCR for ligation of the two DNA fragments. In this connection, approximately 1-5 ng of DNA was used. For ligation of the TVHb-URA DNA fragment, two-step PCR was performed as follows.

In PCR step 1, the TVHb and URA DNAs mixed at a molar ratio of 1 : 1 were mixed with a PCR composition without addition of the primers, and PCR was performed. 1.0 ΐ, of a template DNA (TVHb & URA DNAs, xlOO diluted), 0.2 μΐ of dNTP mix (200 μΜ each), 4.0 μΐ. of 5x iProof HF buffer, 14.6 μΐ, of sterile HPLC water and 0.2 μί of iProof HF DNA polymerase (0.02 \ /μΙ, final) were used as a PCR composition. PCR was performed at 98°C for 5 minutes, followed by 15 cycles of at 98°C for 30 seconds, at 50°C for 30 seconds, and at 72°C for 20 seconds, and then at 72°C for 5 minutes.

In next PCR step 2, the PCR composition of Step 1 was further mixed with the following PCR composition to give a final volume of 40 μΐ, and then PCR was performed. Each 1.0 μΐ of the primers, VHb-F (0.5 μΜ) and URA-R (0.5 μΜ), 0.2 μΐ, of dNTP mix (200 μΜ each), 4.0 μί of 5x iProof HF buffer, 13.6 μί of sterile HPLC water and 0.2 μΐ, of iProof HF DNA polymerase (0.02 U μί final) were used as a PCR composition. PCR was performed at 98°C for 5 minutes, followed by 30 cycles of at 98°C for 30 seconds, at 55°C for 45 seconds, and at 72°C for 20 seconds, and then at 72°C for 5 minutes.

The TVHb-URA ligated DNA having a length of 2314 bp was amplified and prepared by the above two-step PCR (FIG. 4b), and the amplified DNA was separated by electrophoresis on an agarose gel. The separated DNA having a length of 2314 bp was purified using a Gel Extraction Kit provided by Zymo Research Corp. The purified DNA was used as a template DNA in overlapped PCR for ligation with PmtlU DNA. 5) Ligation of PmtlU-TVHb-URA DNA Fragment

The TVHb-URA DNA fragment prepared by PCR was mixed with the PmtlU DNA fragment at a molar ratio of 1 : 1, and then diluted 100-fold with sterile HPLC water to be used as a template DNA in overlapped PCR for ligation of the two DNA fragments. In this connection, approximately 1~5 ng of DNA was used. For ligation of the PmtlU-TVHb-URA DNA fragment, two-step PCR was performed as follows.

In PCR step 1, the TVHb-URA DNA fragment and the PmtlU DNA fragment mixed at a molar ratio of 1 : 1 were mixed with a PCR composition without addition of the primers, and PCR was performed. 1.0 μΐ, of a template DNA (PmtlU + TVHb-URA, xlOO diluted), 0.2 of dNTP mix (200 μΜ each), 4.0 μΐ of 5x iProof HF buffer, 14.6 μΙ_, of sterile HPLC water and 0.2 μΐ, of iProof HF DNA polymerase (0.02 υ/μί final) were used as a PCR composition. PCR was performed at 98°C for 5 minutes, followed by 15 cycles of at 98 °C for 30 seconds, at 50°C for 30 seconds, and at 72°C for 20 seconds, and then at 72°C for 5 minutes.

In next PCR step 2, the PCR composition of Step 1 was further mixed with the following PCR composition to give a final volume of 40 μΊ_^, and then PCR was performed. Each 1.0 μΐ, of the primers, Pmt-UF (0.5 μΜ) and URA-R (0.5 μΜ), 0.2 μί, of dNTP mix (200 μΜ each), 4.0 μΐ, of 5x iProof HF buffer, 13.6 μΐ of sterile HPLC water and 0.2 μΐ, of iProof HF DNA polymerase (0.02 \51μ\. final) were used as a PCR composition. PCR was performed at 98°C for 5 minutes, followed by 30 cycles of at 98°C for 30 seconds, at 55°C for 45 seconds, and at 72°C for 20 seconds, and then at 72°C for 5 minutes.

The PmtlU-TVHb-URA ligated DNA having a length of 3294 bp was amplified and prepared by the above two-step PCR (FIG. 4c), and the amplified DNA was separated by electrophoresis on an agarose gel. The separated DNA having a length of 3294 bp was purified using a Gel Extraction Kit provided by Zymo Research Corp. The purified DNA was used as a DNA to be inserted at the site of the pmtl gene on the chromosome of S. cerevisiae DG5 strain. The obtained DNA was isolated and purified from the agarose gel, and directly transformed into S. cerevisiae DG5. Prior to the transformation, the PmtlU-TVHb-URA DNA fragment was subjected to PCR using each primer set mentioned in Material & Method, and amplification of three DNA fragments was examined to confirm the presence of each DNA fragment.

3- 3. Preparation of Recombinant Yeast having VHb Expression . Cassette Inserted at Site of pmtl gene

200 ng of the PmtlU-TVHb-URA DNA fragment prepared in Example 3-2 was transformed into S. cerevisiae DG5. The transformation was performed using a Yeast Transformation Kit provided by Zymo Research Corp. The transformed cell was plated on a Yeast Synthetic medium agar plate lacking uracil, and cultured at 30°C for 4 days to induce colony formation.

The recombinant strain, S. cerevisiae DG5 prepared in Example 1 is a URA3 deletion mutant strain. Thus, when the URA3 gene is inserted thereto, it can grow successfully in a synthetic medium lacking uracil. That is, the cell growth suggests that the colony formation is attributed to acquisition of the URA3 gene by homologous recombination of PmtlU-TVHb-URA DNA with the pmtl gene on the chromosome of S. cerevisiae DG5.

The genomic DNA was isolated from the obtained colonies, and used as a template DNA to perform PCR, and the presence of PmtlU-TVHb-URA DNA in the transformed strain was finally confirmed (FIG. 4d).

Example 4. GOX Expression Level of Transformed Strain

4- 1. GOx Activity Assay of Recombinant Strain (Qualitative Analysis)

GOx catalyzes the oxidation of 1 mole of glucose to produce an equal amount (1 mole) of H₂0₂. Therefore, changes in the H₂0₂ concentration were measured to determine the GOx activity of the recombinant strain. 0.2 mM o-dianisidine and 10 μ/ml of HRP (250 U/mg) were added to a sterilized medium (1 M KP0₄, 9.5 mM glucose, agarose 0.7%) to prepare agar plates, and the recombinant yeast was inoculated thereto. After cultivating the recombinant yeast for 1 day at 30°C, colonies surrounded by a clear large brown halo on the Petri dishes were selected (FIG. 6).

Each of the selected colonies was inoculated in YPD liquid media containing Aureobasdin A, and shaking-cultured at 30°C and 250 rpm for 2 days. Each of the culture media was centrifuged at 5000 rpm for 10 minutes to collect the supernatant. GOx activity of each supernatant was measured. The colony showing the highest GOx activity was selected and designated as S. cerevisiae DGVh, and deposited at KCTC (Korean Collection for Type Cultures, Genetic Resources Center, Korea Research Institute of Bioscience and Biotechnology, 111 Gwahangno, Yuseong-gu, Daejeon) under Accession No. KCTC12020BP on Sep. 20, 2011.

The following Table 2 shows comparison of GOx activity between S. cerevisiae DGVh strain and its parental strain (S. cerevisiae DG5). As shown in Table 2, the GOx activity of S. cerevisiae DGVh was approximately two times higher than that of its parental strain S. cerevisiae DG-5. In addition, its cell density was 20% higher than those of other strains.

These results suggest that the VHb gene introduced into S. cerevisiae DGVh was successfully expressed, and therefore oxygen supply to the cells was more enhanced to activate the overall metabolism, and consequently, the cell growth and synthesis of the foreign protein GOX were promoted, resulting in high activity. Thus, S. cerevisiae DGVh can be used for mass-production of GOx by the control of aeration levels and high concentration culture using a fed-batch process.

[Table 2]

4-2. Examination of Molecular Weight and Glycosylation of GOx

In order to examine the molecular weight of GOx produced from S. cerevisiae DGVh, SDS-PAGE was performed. As shown in FIG. 5, S. cerevisiae DGVh was found to have the GOx molecular weight of approximately 100 kD, which is 10% reduction, compared to the previously prepared strains having the GOx molecular weight of approximately 110 kD.

Therefore, it is inferred that insertion of the DNA fragment at the site of pmtl gene in S. cerevisiae DGVh leads to the pmtl gene deletion, resulting in reduced glycosylation due to the pmtl deletion.

4-3. GOx Activity Assay of Recombinant Strain (Quantitative Analysis)

For quantitative analysis of GOx, 0.8 ml of 0.2 mM O-dianisidine (molecular weight: 317.21) in ultra pure water, 10 ug of HRP (250 U/mg), and 9.5 mM D-glucose were mixed with each other. Various concentrations of GOx (0, 50, 100, 250, 500, 1000, 2000 ng/0.1ml) were added thereto. After color development for 20 minutes, the enzymatic activity was stopped by addition of 0.1 ml of 4N H₂S0₄. The color developed was measured by absorbance at 400 ran, and a calibration curve of enzymatic activity was prepared. The results are shown in Table 3.

[Table 3]

Example 5. Establishment of Optimal Conditions for Industrial Production of GOx recombinant strain

5-1. Optimization of Initial Inoculation Concentration

In order to examine the optimal inoculation concentration for GOx production, the strain was cultured at 1%, 5%, 10%, and 15% inoculation concentrations in a YPD based media (10 g/L Yeast Extract, 20 g/L Peptone, 20 g/L Glucose). The cultivation was performed at initial pH 5.6, 200 rpm, and 30°C for 7 days. As a result, the final enzymatic activity was 4.0 U/ml at 1% inoculation concentration, and 30.3 U/ml at 10% inoculation concentration, indicating that the initial inoculation concentration greatly affects the final enzymatic activity (FIG. 8). Therefore, the optimal inoculation concentration was determined as 10%.

5-2. Establishment and Optimization of Medium Composition

In order to examine the optimal medium composition for mass-production of GOx using the S. cerevisiae DGVh strain, the known complex medium YPD was replaced with the semi-defined medium composition widely used in Aerobic Baker's Yeast Fermentation. For modification of the Semi-defined medium composition suitable for the strain of the present invention, the medium composition was optimized according to the type and concentration of particular ingredients such as carbon source, nitrogen source, and yeast extract. The results are shown in Table 5.

[Table 5]

5-3. Optimization of Type and Concentration of Carbon source Glucose, sucrose, fructose, or maltose as a carbon source was added to the semi-defined medium, and the cell growth and GOx content were examined by varying the type and concentration of carbon source. The cultivation was performed at initial pH 5.6, 200 rpm, and 30°C for 7 days. As a result, when sucrose was added as a carbon source, the highest cell growth and GOx activity (26.4 U/ml) were observed. Thus, sucrose was determined as an optimal carbon source (FIG. 9).

Next, in order to examine the optimal sucrose concentration, the strain was cultured in the semi-defined medium with a sucrose concentration of 10 g/1, 30 g/1, or 50 g/1. The cultivation was performed at initial pH 5.6, 200 rpm, and 30°C. As a result, when 30 g/1 of sucrose was added, the highest cell growth rate and enzyme activity were observed, and the final enzyme activity was 38.3 U/ml. Upon addition of 1% sucrose, the final enzyme activity was reduced to 31.4 U/ml. In addition, when 50 g/1 of sucrose was added, the final enzyme activity was 28.9 U/ml, indicating that the increasing concentration of the carbon source induces a rapid pH drop and an increase of inhibiting substance (ethanol) at exponential growth phase, leading to inhibition of enzyme production. Therefore, the optimal sucrose concentration was determined as 30 g/1.

5-4. Optimization of Nitrogeii Source Concentration

. In order to examine the optimal concentration of nitrogen source based on the optimal sucrose concentration of 30 g/1, the strain was cultured in the semi-defined medium with a yeast extract concentration of 10 g/1, 20 g/1, 30 g/1, or 40 g/1. The cultivation was performed at initial pH 5.6, 200 rpm, and 30°C. As a result, when 20 g/1 of yeast extract was added, the highest cell growth rate and enzyme activity were observed, and the final enzyme activity was 43.6 U/ml. The enzyme production was reduced at a low concentration of yeast extract. When 40 g/1 of yeast extract was added, the final enzyme activity was 42.8 U/ml. That is, when the enzyme activities were compared at nitrogen source concentrations of 20 g/1, 30 g/1 and 40 g/1, there was little difference in the final cell growth or enzyme activity. Therefore, the optimal concentration of yeast extract was determined as 20 g/1 (FIG. 10).

5-5. Establishment of Culture Conditions in 5 L-Fermentor (Agitation

Speed)

In order to examine the effect of agitation speed during cultivation, the cultivation was performed under the optimal conditions of carbon source (sucrose concentration of 30 g/1) and nitrogen source (yeast extract of 30 g/1) by varying the agitation speed (100 rpm, 200 rpm, 300 rpm, 400 rpm) at 30°C and 1 vvm. As a result, when absorbance at 200 rpm and 300rpm and final enzyme activity were compared, there was little difference in the cell growth, but the final enzyme activity was 42.8 U/ml at 200 rpm, which is higher than 40.1 U/ml at 300 rpm. The enzyme activity was also reduced at the agitation speed of 400 rpm, because of high shear stress and cell damage. Therefore, the optimal agitation speed was determined as 200 rpm (FIG. 11). 5-6. Optimization of Aeration

In order to examine the effect of aeration during cultivation, the cultivation was performed under the optimal conditions of carbon source (sucrose concentration of 30 g/1) and nitrogen source (yeast extract of 30 g/1) by varying the aeration level (0.5 vvm, 1.0 vvm, 1.5 wm). As a result, the final enzyme activity was similarly 44.8 U/ml and 45.2 U/ml at 1.0 wm and 1.5 vvm, respectively, suggesting that the rate of oxygen consumption exceeds the rate of oxygen supply at high cell concentrations, and thus cell growth is restricted. There was little difference in the cell growth between aeration levels of 1.0 wm and 1.5 wm. Therefore, the optimal oxygen supply was determined as 1.0 vvm (FIG. 12).

[Deposit Number]

Depository Institution: Korea Research Institute of Bioscience and Biotechnology

Deposit Number: KCTC 12020BP Deposit date: 20110920

Claims

[CLAIMS]

[Claim 1]

A recombinant vector comprising a glucose oxidase gene of SEQ ID NO. 1.

[Claim 2]

The recombinant vector according to claim 1, further comprising a GPD promoter, an MFa (alpha-mating factor) signal sequence, and a GPD terminator.

[Claim 3]

The recombinant vector according to claim 1, having a cleavage map of FIG. 1.

[Claim 4]

A recombinant yeast transformed with a recombinant vector comprising a glucose oxidase gene of SEQ ID NO. 1.

[Claim 5]

The recombinant yeast according to claim 4, wherein the recombinant vector further comprises a GPD promoter, an MFa (alpha-mating factor) signal sequence, and a GPD terminator.

[Claim 6]

The recombinant yeast according to claim 4, wherein the recombinant vector has a cleavage map of FIG. 1.

[Claim 7]

The recombinant yeast according to claim 4, further comprising a bacterial hemoglobin gene.

[Claim 8]

The recombinant yeast according to claim 7, wherein the bacterial hemoglobin is a Vitreoscilla hemoglobin.

[Claim 9]

The recombinant yeast according to claim 8, wherein the Vitreoscilla hemoglobin gene is inserted at the site of a pmtl gene on the yeast chromosome.

[Claim 10]

The recombinant yeast according to claim 7, wherein the recombinant yeast is further transformed with a recombinant vector comprising a VitreosciUa hemoglobin gene.

[Claim 11]

The recombinant yeast according to claim 10, wherein the recombinant vector comprising a VitreosciUa hemoglobin gene includes a cassette consisting of a TEFl promoter, a VitreosciUa hemoglobin gene, and a CYC ITT terminator.

[Claim 12]

The recombinant yeast according to claim 10, wherein the recombinant vector comprising a VitreosciUa hemoglobin gene includes a first region homologous to the pmtl gene, a TEFl promoter, a VitreosciUa hemoglobin gene, a CYC ITT terminator, a selection marker gene, and a second region homologous to the pmtl gene.

[Claim 13]

The recombinant yeast according to claim 7, wherein the recombinant yeast is Saccharomyces cerevisiae KCTC12020BP.

[Claim 14]

A method for producing glucose oxidase, comprising the step of culturing the recombinant yeast of any one of claims 4 to 13.

[Claim 15]

The method according to claim 14, wherein the recombinant yeast is cultured under any one of the following culture conditions:

(a) 10 to 20% of initial inoculation concentration,

(b) 20 to 40 g 1 of sucrose as a carbon source,

(c) 20 to 40 g/1 of yeast extract as a nitrogen source,

(d) 200 to 400 rpm of agitation speed in a fermenter,

(e) 1.0 to 1.6 vvm of oxygen feed during cultivation, or

(f) at pH 6 to 7, and temperature of 20 to 50°C.

[Claim 16]

A method for producing organic acid calcium using the glucose oxidase produced by the method of claim 14.

[Claim 17]

A composition for the prevention and treatment of metabolic bone disease, comprising the organic acid calcium produced by the method of claim 16.

[Claim 18]

The composition according to claim 17, wherein the metabolic bone disease is osteoporosis or arthritis.

[Claim 19]

The composition according to claim 17, further comprising beta glucan.