JP2007151496A - Method for producing ubiquinone-10 by gene recombination - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a decaprenyl diphosphate synthase gene functioning singly in an yeast, Saccharomyces cerevisiae and capable of producing ubiquinone-10. <P>SOLUTION: The genes encode the following protein (a) or (b). (a) A protein consisting of a specific amino acid sequence derived from Phaffia rhodozyma. (b) A protein composed of an amino acid sequence wherein one or several amino acid(s) are deleted, substituted, or added and also having the decaprenyl diphosphate synthase activity. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a novel decaprenyl diphosphate synthase gene and a method for producing ubiquinone-10 by introducing the gene into a microbial host such as yeast Saccharomyces cerevisiae.

  Ubiquinone exists in the living world from microorganisms to higher animals, and plays a role as a coenzyme in the electron transport system (respiratory chain) in the inner mitochondrial membrane of cells. ). Ubiquinone is composed of a benzoquinone skeleton and isoprene side chains supplied by a prenyl diphosphate synthase (prenyl transferase) that chain-extends isoprene units having 5 carbon atoms in series. The number of repeating isoprene units (n) constituting the isoprene side chain is specific to an organism, for example, 10 in humans, 9 in mice, 8 in E. coli, 7 in Hansenula yeast, and in Saccharomyces yeasts. 6 is known. Ubiquinone-10 (Coenzyme Q-10), which has 10 isoprene units in the human body, is mainly found in cells of organs that require a large amount of energy such as heart, kidney, liver, muscle, and brain. Decreases due to fatigue, decreased immunity, etc. In addition to energy production, ubiquinone-10 is known to significantly inhibit lipid peroxidation and DNA oxidation / damage as an antioxidant. Therefore, in recent years, ubiquinone-10 has been used in various functional foods and cosmetics for the purpose of preventing aging in addition to drugs for treating heart diseases, and has a high market value, and its demand tends to increase. Therefore, a system for producing ubiquinone-10 in large quantities is desired.

  Until now, semi-synthetic methods and microbial fermentation methods have been used for the production of ubiquinone-10. In the production of ubiquinone-10, a decaprenyl diphosphate synthase (DPS) gene involved in the synthesis reaction of its precursor decaprenyl diphosphate (DPP) is a key enzyme.

  So far, Paracoccus denitrificans (Patent Document 1), Rhodobacter capsulatus (Patent Document 2), Schizosaccharomyces pombe (Patent Document 3), Gluconobacter suboxydans (Patent Document 4), Bulleromyces albus (Patent Document 5), Aspergillus genus Alternatively, decaprenyl diphosphate synthase genes derived from various microorganisms such as those derived from the genus Leucosporidium (Patent Document 6) and Saitoella complicata (Patent Document 7) have been isolated. Microbiology of ubiquinone-10 using Escherichia coli as a host Production has been attempted, but productivity is not sufficient. In addition, enzyme genes involved in various carotenoid biosynthetic pathways have been isolated from Phaffia rhodozyma (Patent Document 8), but there are no reports on decaprenyl diphosphate synthase genes.

  On the other hand, when yeast is used as a host, Saccharomyces cerevisiae can only produce ubiquinone-6, and in order to produce ubiquinone-10, decaprenyl diphosphate (DPP) that can supply an isoprene side chain having 10 isoprene units. It is necessary to introduce a decaprenyl diphosphate synthase gene involved in the synthesis reaction. However, for example, the decaprenyl diphosphate synthase gene derived from Paracoccus denitrificans does not function in the yeast Saccharomyces cerevisiae. Therefore, in order to express the gene in the yeast Saccharomyces cerevisiae, the mitochondrial translocation signal sequence is artificially associated with It is necessary to add to a gene (patent document 3). However, such a fusion gene may not only be complicated to construct, but may also lead to a decrease in the inherent enzyme activity.

JP 11-178590 A JP-A-11-56372 Japanese Patent Application Laid-Open No.9-173076 JP-A-10-57072 JP 2002-345469 A JP 2002-191367 A JP 2001-61478 A Special table 2000-507087

  An object of the present invention is to provide a decaprenyl diphosphate synthase gene that functions alone in the yeast Saccharomyces cerevisiae and can produce ubiquinone-10.

  As a result of intensive studies to solve the above problems, the present inventors have focused on the fact that yeast Phaffia rhodozyma produces ubiquinone-10 unlike yeast Saccharomyces cerevisiae. Considering the presence of the decaprenyl diphosphate synthase gene and attempting to clone it, we succeeded in obtaining the gene. Further, when this decaprenyl diphosphate synthase gene was expressed in yeast Saccharomyces cerevisiae, it functioned alone without artificial addition of a mitochondrial translocation signal, and ubiquinone-10 in yeast Saccharomyces cerevisiae into which the gene was introduced. Production was also confirmed. The present invention has been completed based on such findings.

That is, the present invention includes the following inventions.
(1) A gene encoding the following protein (a) or (b).
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing
(b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing and having decaprenyl diphosphate synthase activity
(2) A gene comprising the following DNA (c) or (d):
(c) DNA comprising the base sequence represented by SEQ ID NO: 1 in the sequence listing
(d) a protein that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence represented by SEQ ID NO: 1 in the sequence listing and having decaprenyl diphosphate synthase activity DNA to encode
(3) The following protein (a) or (b):
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing
(b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing and having decaprenyl diphosphate synthase activity
(4) A recombinant vector containing the gene according to (1) or (2).
(5) A transformant transformed with the gene according to (1) or (2).
(6) The transformant according to (5), wherein the transformant is yeast.
(7) The transformant according to (6), wherein the yeast is Saccharomyces cerevisiae.
(8) The transformant according to (6), wherein the yeast is Phaffia rhodozyma.
(9) Production of ubiquinone-10, wherein the transformant according to any one of (5) to (8) is cultured in a medium, and ubiquinone-10 produced and accumulated in the culture is collected. Method.

  According to the present invention, a novel decaprenyl diphosphate synthase gene derived from the yeast Phaffia rhodozyma is provided. The decaprenyl diphosphate synthase gene of the present invention functions in yeast Saccharomyces cerevisiae alone without artificial addition of yeast mitochondrial translocation signal, and efficiently and simply synthesizes ubiquinone-10 used in pharmaceuticals and the like can do.

1. Decaprenyl diphosphate synthase gene The gene encoding the decaprenyl diphosphate synthase (DPS gene) of the present invention (hereinafter referred to as DPS gene) is, first, a known fungal decaprenyl, as shown in Examples below. Comparing gene sequences encoding diphosphate synthase, synthesizing various primers for highly homologous regions, and amplifying the chromosome genome of yeast Phaffia rhodozyma by PCR using various combinations of these primers Thus, a partial gene fragment of the target gene is obtained.

  Next, in order to obtain a full-length gene, the 5 ′ end side base sequence and the 3 ′ end side base sequence are determined by the 5 ′ RACE method and the 3 ′ RACE method, respectively. Specifically, the RACE method is a commercially available primer designed and created based on the internal sequence of the partial gene fragment described above, using mRNA prepared by standard methods from the cells of yeast Phaffia rhodozyma as a template. PCR reaction is performed using the universal primer attached to the RACE cDNA amplification kit (for example, SMART-RACE cDNA Amplification Kit (Clontech)).

  The base sequence of the obtained gene can be determined by a known technique such as a chemical modification method of Maxam-Gilbert or a dideoxynucleotide chain termination method using M13 phage. Usually, an automatic base sequencer (for example, PERKIN- ELMER 373A DNA sequencer, TAKARA BcaBEST dideoxy sequencing kit, etc.). The determined base sequence can be analyzed by DNA analysis software such as DNASIS (Hitachi Software Engineering Co., Ltd.), and the protein coding portion encoded in the obtained DNA strand can be found.

  The DPS gene of the present invention is a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2.

  The gene of the present invention encodes a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having decaprenyl diphosphate synthase activity. Gene to be included.

  Here, the range of “one or several” which may be deleted, substituted or added is not particularly limited. For example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, More preferably, it means 1 to 5, particularly preferably about 1 to 3.

  The gene of the present invention also includes a gene encoding a protein having an amino acid sequence having 70% or more homology with the amino acid sequence shown in SEQ ID NO: 2 and having decaprenyl diphosphate synthase activity. The homology of 70% or more preferably means 80% or more, more preferably 90% or more, and most preferably 95% or more.

  The amino acid deletion, addition and substitution can be performed by modifying the gene encoding the above protein by a technique known in the art. Mutation can be introduced into a gene by a known method such as the Kunkel method or the Gapped duplex method or a method equivalent thereto, for example, a mutation introduction kit (for example, Mutant-K using site-directed mutagenesis). (TAKARA) or Mutant-G (TAKARA)) or the like, or the TAKARA LA PCR in vitro Mutagenesis series kit is used to introduce mutations.

  The above-mentioned “decaprenyl diphosphate synthase activity” means that 7 molecules of isopentenyl diphosphate (IPP, C5) is condensed into E type to farnesyl diphosphate (FPP, C15), and the ubiquinone-10 prenyl side This refers to the activity of synthesizing decaprenyl diphosphate (DPP, C50) as the base of the chain, and “having decaprenyl diphosphate synthase activity” has the amino acid sequence shown in SEQ ID NO: 2 It means that it is substantially equivalent to the activity of protein.

The substantially equivalent activity means that decaprenyl dilin is obtained in a yield of 40% or more, preferably 60% or more, more preferably 80% or more when a protein having the amino acid sequence shown in SEQ ID NO: 2 is used. The ability to synthesize acids. In this activity measurement, farnesyl diphosphate (FPP) and ¹⁴ C-IPP (isopentenyl diphosphate) were reacted with the target enzyme, and the produced ¹⁴ C-DPP (decaprenyl diphosphate) was hydrolyzed by phosphatase, Separation by TLC and confirmation of incorporation into spots of each chain length can be performed (Okada et al., Eur. J. Biochem., 255, 52-59).

  The gene of the present invention also hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence represented by SEQ ID NO: 1 in the sequence listing and has decaprenyl diphosphate synthase activity. Contains DNA encoding the protein.

  Here, stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, it has a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more with a highly homologous nucleic acid, that is, the base sequence represented by SEQ ID NO: 1. Examples include conditions in which complementary strands of DNA consisting of a base sequence hybridize and complementary strands of nucleic acids with lower homology do not hybridize. More specifically, the sodium concentration is 150 to 900 mM, preferably 600 to 900 mM, and the temperature is 60 to 68 ° C, preferably 65 ° C.

  Once the base sequence of the gene of the present invention has been determined, the DNA sequence is then synthesized by chemical synthesis, by PCR using the cDNA of the gene as a template, or by hybridization with a DNA fragment having the base sequence as a probe. The gene of the invention can be obtained.

  The present invention also includes a protein that is an expression product of the DPS gene (hereinafter referred to as “DPS protein”). The DPS protein of the present invention refers to a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing. The DPS protein of the present invention comprises an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, and decaprenyl diphosphate synthase. Proteins having activity are also included. The range of “one or several” is the same as above.

  The DPS protein of the present invention further includes a protein having homology with the amino acid sequence represented by SEQ ID NO: 2. The protein having homology is a protein having homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more with the amino acid sequence represented by SEQ ID NO: 2. Means. To determine protein homology, algorithms described in the literature (Wilbur, W.J. and Lipman, D.J., Proc. Natl. Acad., Sci. USA (1983) 80, 726-730) may be used.

  The DPS protein of the present invention can also be obtained by extracting or separating yeast Phaffia rhodozyma expressing the protein from cultured cells, or by culturing a transformant containing DNA encoding the protein. Can be manufactured.

2. Production of recombinant vectors and transformants
(1) Preparation of recombinant vector The recombinant vector of the present invention can be obtained by linking the DPS gene of the present invention to an appropriate vector. The vector for inserting the DPS gene of the present invention is not particularly limited as long as it can replicate in the host, and examples thereof include plasmid DNA and phage DNA. Examples of plasmid DNA include yeast-derived plasmids (eg, YEp13, YEp24, YCp50, etc.), E. coli-derived plasmids (eg, pBR322, pUC118, etc.), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, etc.), and the like. Examples of DNA include λ phage (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.).

  In order to insert the gene of the present invention into a vector, first, the purified DNA is cleaved with a suitable restriction enzyme, inserted into a restriction enzyme site or a multicloning site of a suitable vector DNA, and linked to the vector. Adopted.

  The vector contains a replication origin, a selection marker, and a promoter, and may contain an enhancer, terminator, ribosome binding site, polyadenylation signal, and the like as necessary.

  As the replication origin, for yeast vectors, for example, those derived from 2 μm DNA, ARS1, for E. coli, for example, those derived from ColE1, R factor, F factor, for animal cells, SV40, Those derived from adenovirus are used.

  Examples of promoters include gal1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter, etc. for yeast vectors, and T7 promoter, trp promoter, lac promoter, PL promoter, PR promoter for E. coli vectors. However, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, etc. are used for animal cell vectors.

  As a selection marker, yeast vectors include auxotrophic marker genes such as His3, Ade2, Lys2, Leu2, Trp1, and Ura3 genes, drug resistance genes such as aureobasidin, cerulenin, canavanine, zeocin, cycloheximide, and tetracycline. However, kanamycin resistance gene, ampicillin resistance gene, tetracycline resistance gene, etc. are used for E. coli vectors, and neomycin resistance gene, thymidine kinase gene, dihydrofolate reductase gene, etc. are used for animal cell vectors.

  Commercially available vectors can be used. For such vectors, when the host cell is yeast, for example, pESP-1 expression vector (STRATAGENE), pAUR123 vector (Takara Shuzo) ), PPIC vector (manufactured by Invitrogen), pYES2 vector (manufactured by Invitrogen), pRS vector (manufactured by STRATAGENE), etc., when the host cell is Escherichia coli, for example, pET vector (manufactured by Novagen), pTrxFUS vector ( Invitrogen), pCYB vector (NEW ENGLAMD Bio Labs), etc., and when the host cell is an animal cell, for example, pMAM-neo expression vector (CLONTECH), pCDNA3.1 vector (Invitrogen) PBK-CMV vector (manufactured by STRATAGENE) and the like.

(2) Production of transformant The transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host so that the target gene can be expressed. Here, the host is not particularly limited as long as it can express the DNA of the present invention, and any of yeast, bacteria, and animal cells may be used, but yeast is preferred. Examples of the yeast include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Phaffia rhodozyma and the like. Examples of bacteria include Escherichia such as Escherichia coli, Bacillus such as Bacillus subtilis, Pseudomonas such as Pseudomonas putida, and Rhizobium meliloti. Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells, human GH3, and human FL cells.

  Among the above hosts, Saccharomyces cerevisiae is preferable, and yeast Saccharomyces transformed by introducing the gene of the present invention is converted so that ubiquinone-10 which is not originally produced can be produced. Phaffia rhodozyma is also preferred, and yeast Phaffia rhodozyma transformed by introducing the gene of the present invention can further enhance the ability to produce ubiquinone-10 inherently.

  When yeast is used as a host, the method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast. For example, electroporation (Becker, DM et al. (1990) Methods Enzymol., 194,182-187), spheroplast method (Hinnen, A. et al. (1978) Proc. Natl. Acad. Sci., USA 75, 1929-1933), lithium acetate method (Itoh, H. ( 1983) J. Bacteriol. 153, 163-168).

  When a bacterium such as Escherichia coli is used as a host, the method for introducing a recombinant vector into the bacterium is not particularly limited as long as it is a method for introducing DNA into the bacterium. For example, a method using calcium ions (Cohen, SN et al. (1972) Proc. Natl. Acad. Sci., USA 69, 2110-2114), electroporation method and the like.

  When animal cells are used as hosts, examples of methods for introducing recombinant vectors into animal cells include electroporation, calcium phosphate, and lipofection.

3. Production of Ubiquinone-10 Ubiquinone-10 can be produced by culturing the above-described transformant in a medium and collecting ubiquinone-10 produced and accumulated from the culture. Here, the “culture” means any of cultured cells or cultured cells or crushed cells or cells in addition to the culture supernatant.

  The method of culturing the transformant of the present invention in a medium can be carried out according to a usual method used for culturing the host cell.

  As a medium for cultivating transformants obtained using microorganisms such as yeast and Escherichia coli as a host, the medium contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the microorganisms, so that the transformants can be cultured efficiently. As long as the medium can be used, either a natural medium or a synthetic medium may be used. Any carbon source may be used as long as the organism can assimilate, and carbohydrates such as glucose, fructose, sucrose, and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used. As the nitrogen source, ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor and the like are used. As the inorganic salts, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used. Depending on the type of selectable marker, antibiotics such as aureobasidin, ampicillin, and tetracycline can be added to the medium, or genes that complement auxotrophy (Leu, Ura, Trp, etc.) can be supplied. Excluding amino acids.

  The culture conditions vary depending on the type of strain and medium, but usually under aerobic conditions such as shaking culture or aeration and agitation culture, the culture temperature is 20 to 40 ° C., the pH of the medium is 3.0 to 7.5, and the culture time is 6 to 6 24 hours is enough. The pH is adjusted using an inorganic or organic acid, an alkaline solution, or the like.

  When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with an expression vector using the Lac promoter, culture a microorganism transformed with isopropyl-β-D-thiogalactopyranoside (IPTG) or the like with an expression vector using the trp promoter. Sometimes indoleacetic acid (IAA) or the like may be added to the medium.

As a medium for culturing a transformant obtained using animal cells as a host, generally used RPMI1640 medium, DMEM medium, a medium obtained by adding fetal calf serum or the like to these mediums, or the like is used. The culture is usually performed at 37 ° C. for 1 to 30 days in the presence of 5% CO ₂ . During culture, antibiotics such as kanamycin and penicillin may be added to the medium as necessary.

  After culturing, when ubiquinone-10 is produced in the microbial cells or cells, the target ubiquinone-10 is collected by disrupting the microbial cells or cells. When ubiquinone-10 is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, ubiquinone-10 may be collected by extraction with an organic solvent or the like, and may be isolated and purified using various chromatography or the like as necessary.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

(Example 1) Cloning of polyprenyl diphosphate synthase gene derived from yeast Phaffia rhodozyma
(1) Acquisition of partial gene fragments After crushing Phaffia rhodozyma (ATCC 66270) with a multi-bead shocker (manufactured by Yasui Kikai Co., Ltd.), using the DNeasy Plant Mini Kit (manufactured by QIAGEN), the chromosome genome was obtained according to the method provided by the manufacturer. Prepared.

  The amino acid sequences of known fungi-derived polyprenyl diphosphate synthase genes were compared, and the following PCR primers (SEQ ID NOs: 3 to 12) were designed and synthesized for highly homologous regions. In the following PCR primer sequences, I is inosine, R is A or G, Y is C or T, D is A or T or G, H is A or C or T, N is A or G or C or T is shown.

PCOQ1-de-1-1f (forward): GI YTI GCN GAR ATH GTN GAR ATG (SEQ ID NO: 3)
PCOQ1-de-1-2f (forward): GCI GAR ATH GTN GAR ATG ATH CA (SEQ ID NO: 4)
PCOQ1-de-1-3f (forward): CAI GCI WSI YTI YTN CAY GAY GA (SEQ ID NO: 5)
PCOQ1-de-1-4f (forward): TI YTI CAY GAY GAY GTN ATH GA (SEQ ID NO: 6)
PCOQ1-de-2-1f (forward): GCI AAY YTI GTN GAR GGN GAR TT (SEQ ID NO: 7)
PCOQ1-de-2-2f (forward): TI GTN GAR GGN GAR TTY ATG CA (SEQ ID NO: 8)
PCOQ1-de-2-1r (reverse): AAY TCI CCY TCN ACI ARR TTN GC (SEQ ID NO: 9)
PCOQ1-de-2-2r (reverse): TCI CCY TCN ACI ARR TTN GCD AT (SEQ ID NO: 10)
PCOQ1-de-3-1r (reverse): GCR AAI ARI ACI GGN GCN TGN GC (SEQ ID NO: 11)
PCOQ1-de-3-2r (reverse): GCI ATI CCI ARY TTN ARR TCN GC (SEQ ID NO: 12)

  PCR was performed using the purified chromosome genome as a template and various combinations of the previously synthesized primers. The obtained PCR product of about 250 bp was excised from the gel, purified from agarose gel using MiniElute Gel Extraction Kit (QIAGEN), cloned into pT7 Blue T-Vector (Novagene), and phDPSp / pT7 Got. The base sequence of the cloned DNA was analyzed using Dye Terminator Cycle Sequence Kit, ABI PRIZM 3100 Gene Analyzer (both Applied Biosystems) and gene information processing program GENETYX-WIN Ver.7 (Software Development Co., Ltd.). As a result, a DNA fragment having a base sequence from 621 to 849 of SEQ ID NO: 1 was obtained from Phaffia rhodozyma. Since these DNA fragments were found to have the amino acid sequence “GDFLXLRA” (X is A or G) in a region characteristic of prenyl diphosphate synthase having a long prenyl chain in the translation sequence, decaprenyl diphosphate synthase It was confirmed to be part of the gene.

(2) Acquisition of gene fragment containing 3 'untranslated region Phaffia rhodozyma (ATCC 66270) was crushed with a multi-bead shocker (manufactured by Yasui Kikai Co., Ltd.) and then used with the RNeasy Plant Mini Kit (manufactured by QIAGEN). Total RNA was prepared according to the method provided.

  Using the obtained total RNA as a template, the following primers (SEQ ID NOs: 13 to 16) designed based on the nucleotide sequence of the DNA fragment obtained in (1) as a forward primer, and RNA PCR Kit (AMV) Ver as a reverse primer RT-PCR was performed using the universal primer attached to .3.0 (Takara) according to the method provided by the manufacturer of the kit.

PDPS-3'-1 (forward): TCG CCC GAG GTA GTC G (SEQ ID NO: 13)
PDPS-3'-2 (forward): TTCT TGC TCG GTA GAG C (SEQ ID NO: 14)
PDPS-3'-3 (forward): ACC TTC AGC ACC CAA TC (SEQ ID NO: 15)
PDPS-3'-4 (forward): CGA TGA TGT GAT TGA TGT TTC TC (SEQ ID NO: 16)

  PCR was performed by repeating a cycle of 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, 72 ° C. for 1 minute 30 times after heat treatment at 94 ° C., and analyzed by 2% agarose gel electrophoresis. The obtained approximately 750 bp PCR product was excised from the gel, purified from agarose gel using MiniElute Gel Extraction Kit (QIAGEN), cloned into pT7 Blue T-Vector (Novagene), and 3 'phDPSp / pT7 was obtained. The base sequence of the cloned DNA was analyzed using Dye Terminator Cycle Sequence Kit, ABI PRIZM 3100 Gene Analyzer (both Applied Biosystems) and gene information processing program GENETYX-WIN Ver.7 (Software Development Co., Ltd.). As a result, a DNA fragment having the base sequence of the 3 ′ untranslated region containing the partial gene obtained in (1) was obtained.

(3) Acquisition of gene fragment containing 5 'untranslated region
The following primers (SEQ ID NOs: 17 to 19) designed based on the base sequence of the DNA fragment obtained in (1) as a reverse primer using the total RNA obtained in (2) as a template, and SMART RACE cDNA Amplification Kit as a forward primer RT-PCR was performed using the attached universal primer (BD Bioscience) according to the method provided by the manufacturer of the kit.
PDPS-5'-1 (reverse) GAG AAA CAT CAA TCA CAT CAT CG (SEQ ID NO: 17)
PDPS-5'-2 (reverse) ACC TTG TTG CCG AAA AGA TTG G (SEQ ID NO: 18)
PDPS-5'-3 (reverse) TCT GGC TAG CGC GGC TGA G (SEQ ID NO: 19)

  PCR was performed by repeating a cycle of 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, 72 ° C. for 1 minute 30 times after heat treatment at 94 ° C., and analyzed by 2% agarose gel electrophoresis. The obtained approximately 800 bp PCR product was excised from the gel, purified from an agarose gel using MiniElute Gel Extraction Kit (QIAGEN), cloned into pT7 Blue T-Vector (Novagene), and 5 'phDPSp. / pT7 was obtained. The base sequence of the cloned DNA was analyzed using Dye Terminator Cycle Sequence Kit, ABI PRIZM 3100 Gene Analyzer (both Applied Biosystems) and gene information processing program GENETYX-WIN Ver.7 (Software Development Co., Ltd.). As a result, a DNA fragment having the base sequence of the 5 ′ untranslated region containing the partial gene obtained in (1) was obtained.

(4) Acquisition of full-length gene In order to obtain the full-length decaprenyl diphosphate synthase gene derived from the yeast Phaffia rhodozyma, the following primers (SEQ ID NO: 20) based on the sequences obtained in (2) and (3) above 21) was designed and synthesized.
PDPS-F (NotI) (forward): GAG AAA CAT CAA TCA CAT CAT CG (SEQ ID NO: 20)
PDPS-R (XhoI) (reverse): ACC TTG TTG CCG AAA AGA TTG G (SEQ ID NO: 21)
Using the total RNA obtained in (2) as a template, RT-PCR was performed according to the method provided by the manufacturer using the above primers and RNA PCR Kit (AMV) Ver. 3.0 (Takara).

  PCR was performed by repeating a cycle of 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, 72 ° C. for 1 minute 30 times after heat treatment at 94 ° C., and analyzed by 2% agarose gel electrophoresis. The obtained about 1.2 kb PCR product was excised from the gel, purified from agarose gel using MiniElute Gel Extraction Kit (QIAGEN), cloned into pT7 Blue T-Vector (Novagene), and phDPS. / pT7 was obtained. The base sequence of the cloned DNA was analyzed using Dye Terminator Cycle Sequence Kit, ABI PRIZM 3100 Gene Analyzer (both Applied Biosystems) and gene information processing program GENETYX-WIN Ver.7 (Software Development Co., Ltd.). As a result, a full-length base sequence of the target gene was obtained. Its size is 1398 bp, encodes 465 amino acids (base sequence: SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 2), and showed high homology with eukaryotic decaprenyl diphosphate synthase gene. .

Example 2 Construction of Expression Vector The plasmid phDPS / pT7 obtained in Example 1 was treated with restriction enzymes NotI and XhoI and then inserted into the yeast Saccharomyces cerevisiae expression vector pRS433GAP to prepare phDPS / pRS433GAP. The resulting expression vector is shown in FIG.

(Example 3) Functional analysis of decaprenyl diphosphate synthase gene derived from yeast Phaffia rhodozyma To examine the function of the decaprenyl diphosphate synthase gene derived from yeast Phaffia rhodozyma, phDPS / pRS433GAP obtained in Example 2 was examined. Was introduced into yeast Saccharomyces cerevisiae (hereinafter referred to as Y03Wd strain) in which the hexaprenyl diphosphate synthase gene (YBR003w) was disrupted by homologous recombination using Frozen Yeast Transformation II (manufactured by ZYMO RESERCH), and the YphDPS strain Was made.

  Next, this YphDPS strain (a strain in which phDPS / pRS433GAP was introduced into the Y03Wd strain), a Y03W-433 strain (a strain in which an empty vector was introduced into the Y03Wd strain), a Y2N14907 strain (a Coq2 (N35) -DPS fusion gene into the Y03Wd strain) The glycerol assimilation ability comparison experiment was carried out on the YPD medium and the YPGlycerol medium for the three strains of the introduced strain).

  YPD medium is 20g / L glucose, 20g / L Bacto Pepton, 10g / L Yeast Extraxt, 20g / L Dissolve agar in water, sterilize in autoclave (121 ℃, 20 minutes), add appropriate amount to plastic plate did. YPGlycerol medium is 30g / L glycerol, 20g / L Bacto Pepton, 10g / L Yeast Extraxt, 20g / L Dissolve agar in water, sterilize in autoclave (121 ° C, 20 minutes), add appropriate amount to plastic plate did.

  The comparison experiment result of glycerol utilization ability is shown in FIG. Y03W-433 strain introduced with only empty vector showed a respiratory deficient phenotype that could not grow on YPGlycerol medium (Fig. 2B, 2), whereas YphDPS strain showed a phenotype with recovered respiratory function that could grow on YPGlycerol medium. (FIG. 2B, 1). From this, it was considered that the ubiquinone biosynthesis ability of the Y03Wd strain was restored by the phDPS gene, so that it became possible to grow on the YPGlycerol medium. That is, it was revealed that the phDPS gene can complement the function of YBR003w in the yeast Saccharomyces cerevisiae cells.

Example 4 Production of Ubiquinone-10 Using Recombinant Yeast The obtained recombinant yeast YphDPS strain was shake-cultured in 5 ml of SD-Ura and His medium at 30 ° C. for 72 hours, and then a portion of the culture solution was 100 ml. SG-Ura and His medium (+0.1 mM PHB) were inoculated. The cells were cultured at 30 ° C. for 72 hours, and the bacteria were collected by centrifugation (1500 rpm, 5 minutes).

  The SD-Ura and His medium are 20 g / L glucose, 1.7 g / L Yeast Nitrogen Base without amino acid, 5 g / L ammonium sulfate, 0.6 g / L CMS-Ura-His-Trp-Leu (manufactured by BIO101) ), 100 mg Leucin, 50 mg Tryptophan, 0.013 g / L parahydroxybenzoic acid was dissolved in water, adjusted to neutral pH with NaOH, and then used after sterilization by autoclave (121 ° C., 20 minutes). SG-Ura, His medium (+0.1 mM PHB) medium is 20 g / L galactose, 1.7 g / L Yeast Nitrogen Base without amino acid, 5 g / L ammonium sulfate, 0.6 g / L CMS-Ura-His-Trp- Leu (manufactured by BIO101), 100 mg Leucin, 50 mg Tryptophan, 0.013 g / L parahydroxybenzoic acid was dissolved in water, adjusted to neutral pH with NaOH, and then used after sterilization in an autoclave (121 ° C, 20 minutes) .

  The collected cells are mixed with 2.5 mg of Westase (Takara Shuzo) in 0.5 ml of McIlveine Buffer pH 6.0 (0.1 M citric acid solution and 0.2 M sodium dihydrogen phosphate solution in a mixing ratio of 36.8: 63.2 to adjust the pH. Adjusted to 6.0) and reacted at 37 ° C. for about 1 hour. Thereafter, about 1 g of glass beads (0.5 mm) and 70 μl of 100 μM ubiquinone-9 (internal standard) were added, and crushing (2500 rpm, 2 minutes) with a multi-bead shocker (manufactured by Yasui Machinery Co., Ltd.) was performed. Next, in order to extract the organic substance containing ubiquinone, 6 ml of methanol and 4 ml of pentane were added and vigorously stirred for 2 minutes. The organic layer (upper layer) separated by centrifugation (1500 rpm, 5 minutes) was transferred to a new test tube. 4 ml of pentane was newly added, stirred vigorously for 2 minutes, and the organic layer obtained by centrifugation was transferred to the previous test tube. The obtained organic layer was dried and concentrated in Speed vac Plus SC210A (Savant) or in a fume hood. This was redissolved with 0.5 ml of ethanol / hexane (9: 1) solvent, then subjected to HPLC SERIES 1100 (manufactured by Hewlett-Packard), and ubiquinone was analyzed by measuring absorbance at 275 nm. The HPLC analysis conditions were ethanol as a solvent, and the flow rate was 1 ml / min for the first 2 minutes and 0.5 ml / min thereafter. The column was ODS-UG-3 4.6Dx150 (manufactured by Nomura Chemical Co., Ltd.) and kept at 25 ° C. Analysis was performed on a peak (Restriction time: about 8 minutes) detected at the same position as ubiquinone-10 used as a standard. The production amount of ubiquinone-10 calculated from the extraction efficiency of ubiquinone-9 used as an internal standard was calculated. As a result, production of ubiquinone-10 was not confirmed in the comparative control Y03W-433 strain, whereas production of ubiquinone-10 was confirmed in the YphDPS strain into which the phDPS gene was introduced (FIG. 3).

The structure of the expression vector phDPS / pRS433GAP is shown. The results of the glycerol utilization test (performed three times for each strain) of YphDPS strain, Y03W-433 strain, and Y2N14907 strain are shown (1: YphDPS strain, 2: Y03W-433 strain, 3: Y2N14907 strain). The ubiquinone-10 production amount of YphDPS strain and Y03W-433 strain is shown.

Claims (9)

A gene encoding the following protein (a) or (b).
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing
(b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing and having decaprenyl diphosphate synthase activity A gene comprising the following DNA (c) or (d):
(c) DNA comprising the base sequence represented by SEQ ID NO: 1 in the sequence listing
(d) a protein that hybridizes under stringent conditions with a DNA comprising a nucleotide sequence complementary to the DNA comprising the nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing and has decaprenyl diphosphate synthase activity DNA to encode The following protein (a) or (b).
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing
(b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing and having decaprenyl diphosphate synthase activity   A recombinant vector containing the gene according to claim 1 or 2.   A transformant transformed with the gene according to claim 1 or 2.   The transformant according to claim 5, wherein the transformant is yeast.   The transformant according to claim 6, wherein the yeast is Saccharomyces cerevisiae.   The transformant according to claim 6, wherein the yeast is Phaffia rhodozyma.   A method for producing ubiquinone-10, wherein the transformant according to any one of claims 5 to 8 is cultured in a medium, and ubiquinone-10 produced and accumulated in the culture is collected.

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