JPH1057072A - Production of ubiquinone-10 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new transformed microorganism, transformed with a gene capable of coding a decaprenyl diphosphate synthase(DDS) and capable of producing ubiquirtone-10 useful for treatment, etc., of cardiopathy, myodystrophy and anemia, etc. SOLUTION: This new transformed microorganism such as Escherichia coli JM109/pLD2 or Escherichia coli K0229/pLD2 is transformed with a gene capable of coding a decaprenyl diphosphate synthase(DDS) having an amino acid sequence represented by the formula. The transformed microorganism is capable of producing only high purity ubiquinone-10 effective in treating congestive heat failure, myodystrophy, anemia, etc., when using Escherichia coli deficient in a structural gene ispB of an octaprenyl diphosphate synthase (OPS) as a host. The transformed microorganism is obtained by carrying out a polymerase chain reaction(PCR) of a genomic DNA of an acetic acid bacterium with a primer comprising a fragment of the DDS gene, integrating the resultant DDS gene in a vector and transducing the prepared vector into a host microorganism.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the production of ubiquinone, especially ubiquinone-10. More specifically, the present invention relates to decaprenyl diphosphate synthase (D
DS), which is a novel system for producing ubiquinone-10 by isolating and expressing a gene encoding ubiquinone-10.

[0002]

2. Description of the Related Art Isoprenoids are natural organic compounds having isopentenyl diphosphate (IPP) having 5 carbon atoms as a basic unit, and are present in a large number in nature. Examples include pigment carotenoids, natural rubber, isoprenoid quinones that work in the respiratory chain electron transport system, and the like.
There are several types of isoprenoid quinones, such as ubiquinone (UQ), menaquinone, and plastoquinone, which are distinguished by differences in the quinone skeleton, and each has a large number of homologues due to differences in isoprenoid side chain length. Ubiquinone is a compound in which isoprenoids are added to the quinone skeleton, and 2,3-dimethoxy-5-methyl-6-polyp
It has the structure of renyl-1,4-benzoquinone and has a coenzyme (Co
It is a biological component that plays an important role also called Q). Ubiquinone is a vitamin-like substance known as an antioxidant, which exists as a bacterial component of animal and plant tissues and microorganisms, and plays important physiological and biochemical functions as an essential component of the electron transport system. . In nature, homologues from ubiquinone-1 to ubiquinone-12 mainly exist depending on the number of isoprenoid units in the side chain.

[0003] Ubiquinone has been studied for its pharmacology and clinical effects and is considered to be effective in congestive heart failure, muscular dystrophy, anemia and the like, and is a valuable substance as a drug for heart diseases and other various pharmaceuticals. However, only ubiquinone-10 is effective as a pharmaceutical. Ubiquinone-10 is currently extracted and purified from tobacco leaves, yeast or microbial cells, but the details thereof have not been clarified. However, its production cannot keep up with demand, and there is a need to develop more efficient production methods that can replace conventional methods.

The isoprenoid forming the side chain of ubiquinone is isopentenyl diphosphate having 5 carbon atoms (IP
It is a natural organic compound having P) as a basic unit, and is present in a large number in nature. In addition to being used as the side chain of ubiquinone, it is also known as a carotenoid, natural rubber, and as a prenylated protein. In biosynthesis in a living body, new IPP binds to basic IPP or FPP (funesyl diphosphate), and gradually becomes isoprenoids having a long chain length. Such an enzyme that catalyzes the synthesis of isoprenoid is called prenyltransferase. Many types of prenyltransferases have been confirmed mainly in bacteria. FPS, OP for E. coli
The existence of three enzymes, S and UPS, has been confirmed, and the genes isolated are the structural genes of FPS, ispA and octaprenyl diphosphate synthase (OPS)
Is the structural gene ispB.

[0005] By the action of this prenyltransferase, octaprenyl diphosphate having a side chain length of 8 is synthesized in Escherichia coli, and hexaprenyl diphosphate having a side chain length of 6 is synthesized in budding yeast. On the other hand, E. coli ubiA product, yeast CO
These isoprenoids are added to the benzoquinone skeleton derived from PHB (p-hydroxybenzoate) by the Q2 product to complete ubiquinone-8 and ubiquinone-6, respectively. From these facts, it is considered that the point at which the ispB product and the ubiA product catalyze is important for ubiquinone synthesis. Ubiquinone-10
Is attracting attention as a physiologically active substance having a high pharmacological action, as described above. With a new perspective that this leads to the production of a product, he focused on the necessity of cloning the gene for decaprenyl diphosphate synthase.

[0006]

DISCLOSURE OF THE INVENTION In view of the above-mentioned state of the art, the present invention develops a technique for producing UQ-10 in a microorganism using a gene involved in the synthesis of decaprenoid, which is a side chain of UQ-10. It was done for the purpose.

[0007]

DISCLOSURE OF THE INVENTION The present invention has been made in order to achieve the above-mentioned object, and as a result of investigations from various fields, it has been found that a biologically active organism producing UQ-10 can be synthesized from the organism. The necessary gene, that is, the gene for decaprenyl diphosphate synthase necessary for synthesizing the side chain decaprenyl diphosphate, was isolated and expressed in Escherichia coli to produce UQ-10.

[0008] To solve this new technical problem, UQ-
Acetobacter bacterium Gluconobacter subo
PCR was performed using xydans genomic DNA as a template. The primers used at that time were designed with reference to the region highly conserved in polyprenyl diphosphate synthase.
xed primer was used. The fragment obtained by this PCR was cloned into a vector, the nucleotide sequence was determined, and those having similar predicted amino acids to those of known polyprenyl diphosphate synthase were selected. Thereafter, genomic Southern hybridization was performed on the G. suboxydans genome using the obtained fragment as a probe. Based on the information obtained, a partial genome library was prepared, and a gene region including the entire region of this gene was obtained by colony lift assay. After determining the nucleotide sequence of the region that would encode prenyl diphosphate synthase in this gene region, orf was newly amplified by PCR, cloned into an E. coli expression vector, and the promoter of E. coli lacZ gene was used. Expression. UQ was prepared from Escherichia coli expressing this gene,
C confirmed. In addition, enzyme activity was measured using a crude enzyme protein prepared from similar cells.

In addition, Escherichia coli deficient in the octaprenyl diphosphate synthase gene (ispB gene) required for UQ-8 synthesis has been prepared, while the dds1 gene has been added to the E. coli expression vector in the same manner as described above. It was also confirmed that UQ-10 was generated as a main product by cloning and expressing this in the above-mentioned ispB-disrupted strain.

As a result, UQ-10 was successfully manufactured for the first time. And in that case, in the former case,
UQ-10 was successfully produced together with UQ-8 and UQ-9, and in the latter case using an ispB-disrupted strain, UQ-10 was mainly produced, and high-purity UQ-10 was produced. It was successfully manufactured efficiently, and based on these successes, the present invention was completed. that's all,
The present invention will be described in detail.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION

(1) Synthesis of two types of oligo primers shown in SEQ ID NO: 4 (sense primer) and SEQ ID NO: 5 (antisense primer) in the sequence listing, and several times
CR was performed. That is, 95 ° C. for 1 minute, 55 ° C. 2
The initial PCR was performed for 30 minutes at 72 ° C. for 1 minute, and the vicinity of 400 to 500 b was cut out by agarose electrophoresis, and the same reaction was performed again. As a result, about 40
A main band was obtained around 0b. This PCR fragment was directly cloned into a vector and the nucleotide sequence was determined. As a result, three types of sequences were determined. When the amino acid sequence of all of them was compared with the existing prenyltransferase, one of them had about 40% homology with E. coli octaprenyl diphosphate synthase. Therefore, this fragment was considered to be a part of the decaprenyl diphosphate synthase gene of G. suboxydans, and the entire region was screened as follows.

(2) First, using this fragment as a probe,
Southern hybridization was performed on the genome of suboxydans. As a result, when a genomic EcoRI digest was used, a signal was obtained at about 5 kb. After genomic EcoRI digestion, about 5 kb was cut out by agarose electrophoresis and ligated to the same site of the vector to obtain a partial genomic library. did. Thereafter, colony hybridization was performed on Escherichia coli holding this library using the above PCR fragment as a probe. As a result of screening about 6000 transformants, several positive colonies were obtained. A plasmid was prepared from the cells and subjected to Southern hybridization. As a result, a plasmid that could be determined to be positive was obtained, and was named pGS1.

(3) Since the plasmid pGS1 has a fragment of about 5 kb, the Sal1I-Hind
III A nucleotide sequence of about 2.1 kb was determined (FIG. 1). An ORF (open reading frame), which is a portion where the genetic information of DNA is transcribed into m-RNA and translated into protein, was searched from the sequenced DNA sequence. In the DNA codon, the transcription initiation portion of the m-RNA is A
Since the TG and the transcription termination part are any of TAG, TGA and TAA, the start codon and the stop codon are searched.
An ORF determination was made. As a result, an ORF of about 1 kb containing the above PCR fragment was found.

(4) As shown in FIG. 1, this gene
It has an ORF consisting of 948 bp and 315 amino acids which starts from position 808 (ATG: methionine) and ends at position 1756 (TAA: no corresponding amino acid) shown in the nucleotide sequence of FIG. Shows high homology to acid synthase, Escherichia coli IspB, yeast Sa
ccharomyces cerevisiae Coq1, Bacillus su
btilis heptaprenyl diphosphate synthase,
There was 47.9%, 45.3%, 32.4% homology. Therefore, this gene was named decaprenyl diphosphate synthase gene dds1, and its nucleotide sequence is shown in SEQ ID NOs: 1 and 2 together with the amino acid sequence of DDS.

(5) Based on the obtained sequence data, oligo primers (sense primers shown in SEQ ID NO: 4 and antisense primers shown in SEQ ID NO: 5) capable of amplifying the region containing the ORF were synthesized, and the insert DNA of dds1 was synthesized. (Its base sequence is shown in SEQ ID NO: 3)
Amplified with CR. The amplified insert DNA is 5 '
The BamHI site and the HindIII site are stored on the 3 'and 3' sides, respectively.
18 (Takara Shuzo) at the BamHI-HindIII site. By ligating the insert DNA to the above site, dds1 was expressed in a form fused with the lacZ gene of the vector. That is, the OR of dds1
Plasmid pLD2 was constructed to express F under the promoter of E. coli lacZ gene (FIG. 2).

A single colony of Escherichia coli JM109 carrying the plasmid pLD2 was inoculated with 10 ml of an LB liquid medium containing 50 μg / ml of ampicillin using a sterilized toothpick and cultured with shaking at 37 ° C. overnight. 2.5 ml of precultured culture medium in 250 ml of the same medium as above
(1% inoculated), and cultured with shaking at 37 ° C. for 2 to 3 hours. Thereafter, it was confirmed by a spectrophotometer that OD ₆₀₀ was about 0.5, and 1M IPTG (isopropyl-1-
(Thio-β-D-galactoside) was added in an amount of 0.25 ml (final concentration: 1 mM), and the cells were cultured for another 12 hours.
When UQ was prepared from the obtained cells and analyzed by HPLC, as shown in FIG.
Was detected. Peak 1 is UQ-8, but peak 3
Is consistent with UQ-10 of the reference material, so UQ-10
And peak 2 is considered to be UQ-9.

Escherichia coli JM109 (Es) harboring pLD2, which is a transformed microorganism transformed with dds1
cherichia coli JM109 / pLD2) was converted to isopropyl-1-thio-β-D-
Culture was performed in the absence of galactoside (IPTG), and UQ was similarly prepared. As a result, the amount of production was reduced to one-tenth. This also indicates that dd introduced as a plasmid was used.
It has been revealed that UQ-10 can be produced by using the s1 gene.

Next, Escherichia coli JM109 / p
A crude enzyme protein was prepared from LD2, and the activity was measured.
That is, using ¹⁴ C-IPP (impentenyl diphosphate: Amersham LIFESCIENCE) and FPP (farnesyl diphosphate) as substrates, reacting at 30 ° C. for 4 hours, and then extracting polyprenyl diphosphate with butanol. After hydrolysis, it was developed by TLC in the state of polyprenol. As a result, as shown in FIG.
9, decaprenol was detected. From these results, it was revealed that the dds1 gene obtained was a gene responsible for the synthesis of the side chain decaprenyl diphosphate required for UQ-10 synthesis, and by expressing the dds1 gene in E. coli, It has been demonstrated that -10 generation is possible.

(6) As described above, UQ-8 and UQ-9 using Escherichia coli JM109 transformed with plasmid pLD2
The example of the generation of UQ-10 accompanied by the generation of UQ-10 has been described. Next, an example of a system that can generate UQ-10 with a more pure product will be described.

(7) Preparation of KO229 / pLD2 As described below, Escherichia coli
KO229) cannot synthesize UQ-8 because it lacks the ispB gene required for UQ-8 synthesis. Therefore, expression of plasmid pLD2 in this strain can be expected to produce UQ-10 alone as a single product or to increase the amount of UQ-10 produced. In other words, the above-mentioned Escherichia coli JM109
/ PLD2 generates UQ-10, but UQ-8
Is the main product, whereas Escherichia coli KO
229 / pLD2 uses UQ-10 as a main product, and the present example aims at high-purity production of UQ-10.

A) Disruption of ispB Gene on Plasmid First, ispB of plasmid pKA3 carrying ispB
Was digested with a restriction enzyme. The cat (chloramphenicol acetyltransferase) gene was introduced therein. At that time, pKA3, ca
Both sites of t were blunt-ended with T4 polymerase before ligation. The cat gene is p
ACYC184 (Nippon Gene) was prepared by HaeII treatment. The region of the ispB gene separated by cat from the obtained plasmid pTC2 was EcoRI
It was cut out by the treatment, and this was used as fragment Q.

B) Preparation of ispB-deficient strain Escherichia coli FS1576, which is liable to undergo homologous recombination, was transformed into pKA
3 was transformed. The resulting transformant FS1576 /
pKA3 was again transformed with fragment Q prepared in a). The transformants obtained here were chloramphenicol (Cm) and spectinomycin (Spc) in 50 parts.
This strain was grown on an LB agar medium containing μg / ml, and the chromosome or the ispB gene on pKA3 was replaced by fragment Q (homologous recombination had occurred). PKA3 was taken from the obtained Cm + Spc resistant strain, and a strain whose size did not change was selected. In the strain selected here, the ispB gene on the chromosome was disrupted by cat, and instead, the intact ispB gene was retained as the plasmid pKA3. E. coli K
O229 / pKA3.

C) Isolation of KO229 / pLD2 strain The KO229 / pKA3 obtained in b) was transformed with the plasmid pLD2 having the dds1 gene. Since pLD2 has an ampicillin (Amp) resistance gene, Cm, S
Selection was made on an LB agar medium containing 50 μg / ml each of pc and Amp. Obtained KO229 / pKA3 + pLD
Two strains were cultured in an LB liquid medium containing Amp + Cm, and subcultured to a similar new medium when saturated. This operation is repeated a total of five times, and the mixture is diluted on an LB agar medium containing Amp + Cm. Am + Cm resistant strains that grew
Replicas were replicated one by one on p + Cm and Spc + Cm agar medium, and Amp + Cm-resistant, Spc-sensitive strains were selected. The obtained strain was used as E. coli KO229 / pLD2
Used for production of Q-10. In view of the usefulness of this strain, this strain was named Escherichia coli KO229 / pLD2, and FERM BP-5626 was instituted by the National Institute of Bioscience and Biotechnology, National Institute of Advanced Industrial Science and Technology.
As an international deposit.

(8) Production of UQ-10 E. coli JM transformed with pLD2 in the previous example
As in the case of culturing Escherichia coli K.109,
O229 / pLD2 (FERM BP-5626) at 37 ° C. in LB medium containing 50 μg / ml ampicillin
1% of the pre-cultured cells was inoculated into a fresh medium, and when the OD ₆₀₀ reached about 0.5, IPTG was added to 1 mM and further cultured until the late logarithmic growth phase. UQ was prepared from the obtained cells and analyzed by HPLC. As shown in FIG. 5, a main peak of UQ-10 was observed, and KO229 / pLD2 (FERM BP) was observed.
-5626), the production of high-quality UQ-10 was confirmed. In the case of this strain, JM109 / p of the previous example was used.
Unlike in the case of LD2, a slight increase in the production of UQ-10 was observed when the cells were cultured in the IPTG-supplemented medium, but no remarkable difference in the production was observed when cultured in the IPTG-free medium. Was.

[0025]

According to the present invention, not only the structural gene of decaprenyl diphosphate synthase was isolated and successfully sequenced, but also expressed for the first time in Escherichia coli. As a result, in addition to producing UQ-8 and UQ-9,
Not only is it possible to produce Q-10 by E. coli for the first time, but by using an ispB gene-deficient strain,
It has become possible for the first time to selectively produce UQ-10 with high purity and in large quantities by suppressing the production of UQ-8.

[0026]

[Sequence List] Amino acid sequence of DDS according to the present invention (315
Amino acid) is shown in SEQ ID NO: 1, and the nucleotide sequence (948 bp) of the gene (dds1) encoding it is shown in SEQ ID NO: 2.
And the region from the sense primer to the antisense primer containing the dds1 gene is shown in SEQ ID NO: 3.

SEQ ID Nos. 4 and 5 show primers for PCR, SEQ ID No. 4 shows a sense primer, and SEQ ID No. 5 shows an antisense primer. Tables 1 to 10 below show the respective sequences shown in SEQ ID NOs: 1 to 5.

[0028]

[Table 1]

[0029]

[Table 2]

[0030]

[Table 3]

[0031]

[Table 4]

[0032]

[Table 5]

[0033]

[Table 6]

[0034]

[Table 7]

[0035]

[Table 8]

[0036]

[Table 9]

[0037]

[Table 10]

[Brief description of the drawings]

FIG. 1 shows a nucleotide sequence containing a gene encoding DDS.

FIG. 2 shows the expression plasmid pLD2.

FIG. 3 shows an HPLC chromatogram of UQ extracted from E. coli JM109 / pLD2 culture.

FIG. 4 shows a thin-layer chromatogram of E. coli JM109 / pLD2 product.

FIG. 5 shows an HPLC chromatogram of UQ extracted from E. coli KO229 / pLD2 culture.

Continued on the front page (51) Int.Cl. ⁶ Identification number Reference number in the agency FI Technical display location C12R 1:19) (C12P 7/66 C12R 1:19)

Claims (11)

[Claims] 1. Decaprenyl diphosphate synthase (DD)
A transformed microorganism obtained by transformation with a gene encoding S). 2. An octaprenyl diphosphate synthase (OP)
The transformed microorganism according to claim 1, wherein the gene encoding S) is disrupted. 3. The DNA of a gene encoding DDS represented by the amino acid sequence of SEQ ID NO: 1 in the sequence listing. 4. The DNA of the gene encoding DDS (dds1 gene) according to claim 3, which is represented by the nucleotide sequence of SEQ ID NO: 2. 5. A DNA of a gene represented by the nucleotide sequence of SEQ ID NO: 3 and comprising the DNA of the gene encoding the amino acid sequence of SEQ ID NO: 1. 6. A plasmid comprising the DNA of a gene encoding DDS. 7. D according to any one of claims 3 to 5,
Expression plasmid pLD2 obtained by inserting NA into vector pUC18. 8. A transformed microorganism obtained by transforming with the plasmid according to claim 6 or 7. 9. The transformed microorganism is Escherichia coli JM109 /
The transformed microorganism according to claim 8, which is pLD2 or Escherichia coli KO229 / pLD2. 10. A ubiquinone-1 comprising culturing the transformed microorganism according to claim 1, 2, 8 or 9.
Generation method of 0. 11. A method for producing high-purity ubiquinone-10, which comprises culturing Escherichia coli KO229 / pLD2.