JPH09173076A - Method for producing heterogeneous ubiquinone - Google Patents

Abstract

(57) [Summary] [Solution] Saccharomyces cere
A fragment containing the translocation sequence of the structural gene COQ1 of hexaprenyl diphosphate synthase of visiae to mitochondria is fused with a heterologous prenyltransferase gene to prepare a fusion gene. [Effect] By expressing a fusion gene, for example, when the ispB gene is used, octaprenyl diphosphate synthase and when the decaprenyl diphosphate synthase gene is used, the enzyme is heterologously produced in yeast. be able to.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing heterogeneous ubiquinone. More specifically, Saccharom
Yeast cerevisiae hexaprenyl diphosphate synthase structural gene COQ1 is used to express a heterologous prenyl transferase gene to realize the production of the enzyme, the production of isoprenoid, and the production of various ubiquinones in yeast. Is.

[0002]

Ubiquinone is a compound in which an isoprenoid is added to a quinone skeleton, which is 2,3-dimeth.
oxy-5-methyl-6-polyprenyl
It is a biological component that has a structure of -1,4-benzoquinone and plays an important role also called coenzyme (CoQ). Ubiquinone exists as a tissue component of plants and animals, as a bacterial cell component of microorganisms, and fulfills physiologically and biochemically important functions as an essential component of the electron transfer system. Naturally, there are mainly homologues of ubiquinone-6 to ubiquinone-10 depending on the number of isoprenoid units in the side chain.

Ubiquinone has been studied for its pharmacological and clinical effects and is said to be effective for congestive heart failure, muscular dystrophy, anemia and the like, and is a highly valuable substance as a drug. However, only ubiquinone-10 has been confirmed to be effective as a medicine. Ubiquinone-1
0 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 (IP) having 5 carbon atoms.
It is a natural organic compound having P) as a basic unit and exists in large numbers in nature. Besides being used as a side chain of ubiquinone, its appearance as carotenoid, natural rubber, and prenylated protein is also known. In biosynthesis in vivo, new IPP is bound to basic IPP and FPP (fanesyl diphosphate), and gradually becomes an isoprenoid with a long chain length. The enzyme that catalyzes the synthesis of isoprenoids is prenyltransferase. It has been confirmed that prenyltransferases exist in various types, mainly in bacteria. E. FIG. In E. coli, FPS,
The existence of three enzymes, OPS and UPS, has been confirmed,
The genes that have been isolated are FPS structural genes ispA and octaprenyl diphosphate synthase (OP
S) is a structural gene ispB.

By the action of this prenyltransferase, octaprenyl diphosphate having a side chain length of 8 is synthesized in Escherichia coli and hexaprenyl diphosphate is synthesized in budding enzyme. On the other hand, these polyisoprenoids are added to the benzoquinone skeleton derived from PHB (p-hydroxybenzoate) by Escherichia coli ubiA product and yeast COQ2 product, and ubiquinone-8 and ubiquinone-6, respectively.
Is completed. From this, the ispB product and ub
It is considered that the point at which iA products catalyze is important for ubiquinone synthesis. Ubiquinone-10
Is attracting attention as a physiologically active substance with a high pharmacological action, but it is expected to lead to efficient production of ubiquinone-10 by utilizing the gene for decaprenyl diphosphate synthase that synthesizes a side chain and the ubiA product and yeast COQ2 product. From the viewpoint of ubiquinone-6 in E. coli,
Alternatively, attention was paid to the fact that it was necessary to establish a technique for producing ubiquinone-8 in yeast and to clone the decaprenyl diphosphate synthase gene.

[0006]

SUMMARY OF THE INVENTION In view of the above-mentioned state of the art, the present invention has been made for the purpose of developing a technique for producing a heterologous ubiquinone in yeast by utilizing a gene involved in isoprenoid synthesis, which is a side chain of ubiquinone. It is a thing.

[0007]

[Means for Solving the Problems] The present invention has been made in order to achieve the above-mentioned object, and as a result of researches from various fields, the ispB gene encodes 53 amino acids containing a translocation sequence of COQ1 to mitochondria. Succeeding in the creation of a fusion gene that fuses with the region to be expressed, and in the expression of this fusion gene in Saccharomyces cerevisiae, ubiquinone-8 that Escherichia coli retains, not ubiquinone-6 that Saccharomyces cerevisiae normally retains, was synthesized. We succeeded in synthesizing ubiquinone of different species in the organisms for the first time. In addition, they have succeeded in cloning and sequencing the decaprenyl synthase gene of fission yeast, and have completed the present invention based on these successes. Less than,
The present invention will be described in detail.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION

(1) In order to express the budding yeast COQ1 gene in Escherichia coli, an ispB gene-disrupted strain was first prepared. Using the chloramphenicol acetyltransferase gene as a marker gene,
Destroy the pB gene. That is, P of ispB gene
The chloramphenicol acetyltransferase gene (CAT) was inserted into the stI site, and the plasmid pT
Obtain C2. Escherichia coli FS1576 is transformed by cutting out a necessary portion from pTC2. FS1576 is Escherichia coli capable of easily causing homologous recombination with the chromosome, and the strain that has undergone homologous recombination becomes resistant to chloramphenicol.

However, no colony was obtained as a result of the transformation. In such cases, the target i
The spB gene is considered to be essential for growth. Therefore, a plasmid (pKA3) having the ispB gene was held in FS1576 in advance, and homologous recombination was performed in the same manner as above. Then, many chloramphenicol resistant strains could be obtained. Those in which the recombination occurred on the chromosome were selected by checking the plasmid. Chromosomes were prepared for the strains selected in this manner and subjected to genomic Southern hybridization.
Check whether it has been recombined on spB,
The spB-disrupted strain was named KO229. Since KO229 retains pKA3, it is considered that this plasmid complements the deletion on the chromosome.

Therefore, next, an experiment was conducted to drop pKA3 from KO229. In Escherichia coli, the plasmid that is no longer required for growth is not separated into daughter cells during division, and it can be confirmed that the plasmid is dropped. That is, it is subcultured several times in a non-selective medium for the plasmid. Plate on non-selective plates and replicate to selective and non-selective plates to isolate sensitive strains in selective media. However, a strain in which pKA3 had been completely lost could not be isolated.

(2) Saccharomyces cerevisiae as a mitochondrial translocation sequence
Hexaprenyl diphosphate synthase gene (COQ1
Gene), it seems to contain a transition sequence of 53 amino acids and 103 amino acids from the N terminus using PCR method 2
Genes of species length were generated and these were respectively transformed on the shuttle vector YEp13M14 with Escherichi.
The plasmids pYE6 and pYE7 were prepared by fusing with the acoli octaprenyl diphosphate synthase gene (ispB gene). Then, after introducing the plasmid pYE6 into a wild strain SP1 and a COQ1-deficient strain (YKK6) that cannot synthesize ubiquinone, ubiquinone was identified in a glucose medium, and only ubiquinone-6 production was confirmed (FIG. 1).

(3) In the above, ubiquinone was extracted in a glucose medium, whereas ubiquinone was identified after culturing in a glycerol medium which is a respiratory substrate. Results of HPLC analysis after extraction of ubiquinone (Fig. 2),
It was revealed that YKK6, which normally does not synthesize ubiquinone, synthesizes ubiquinone-8 by retaining pYE, and that those who retain the COQ1 gene synthesize ubiquinone-6. In addition, both ubiquinone-6 and ubiquinone-8 were produced when pYE6 was retained in SP1 which is a wild strain of Saccharomyces cerevisiae. These results represent the first successful synthesis of a different type of ubiquinone in other organisms, indicating that the side chain of ubiquinone is determined by prenyl transferase. Further, it was revealed that ubiquinone-8 functionally complements ubiquinone-6 in Saccharomyces cerevisiae.

(4) In order to clarify whether or not the prenyltransferase in the budding yeast YKK6 / pYE6 producing ubiquinone-8 is synthesizing octaprenyl diphosphate having 8 units, the product is identified. went. The prenyl transferase prepared from YKK6 / pYE6 was reacted with the substrate ¹⁴ C-labeled IPP and unlabeled FPP, and the extended product was developed by TLC. As a result, in normal phase, reverse translocation was found at the same position as solanesol in all trans.
In d phase, a signal was obtained at a position slightly smaller than that of C45 solanesol. From these things, YK
The isoprenoid synthesized by K6 / pYE6 is all-trans octaprenyl diphosphate, and it was revealed that the ispB gene was reliably expressed in Saccharomyces cerevisiae (FIG. 3).

(5) As is clear from the above, S. c
It was confirmed that the migration to mitochondria was sufficient in the 53 amino acid region on the N-terminal side of hexaprenyl diphosphate synthase in Erevisiae, but this was also confirmed from the following.

That is, S. coli was isolated using octaprenyl diphosphate synthase of E. coli. Three plasmids were constructed in order to carry out a complementation experiment of the hexaprenyl diphosphate synthase deficient strain of S. cerevisiae. The first one is octaprenyl diphosphate synthase linked to the ADH promoter, the second is octaprenyl diphosphate synthase linked to the N-terminal 53 amino acids of hexaprenyl diphosphate synthase, and the third is A structure in which octaprenyl diphosphate synthase was connected to 103 amino acids on the N-terminal side of hexaprenyl diphosphate synthase was constructed. It is believed that there is a mitochondrial translocation signal on the N-terminal side of hexaprenyl diphosphate synthase. In addition, S.I. cerev
Hexaprenyl diphosphate of isiae is destroyed by URA3. When the three constructed plasmids were introduced into the hexaprenyl diphosphate synthase disrupted strain, only those with the addition of 53 amino acids complemented the hexaprenyl diphosphate synthase deficiency (FIG. 4). As a result of measuring the activity of prenyl diphosphate synthase, S. cerevisiae
In the hexaprenyl diphosphate synthase-deficient strain of S. cerevisiae, the one obtained by adding octaprenyl diphosphate synthase to which 53 amino acids at the N-terminal side of hexaprenyl diphosphate synthase was added was expressed by S. It was confirmed that octaprenyl diphosphate was synthesized in S. cerevisiae.

(6) From the above results, it was suggested that the ispB gene is essential for the growth of Escherichia coli, and the following was also clarified.

53a. Containing a sequence for translocation of Coq1 to mitochondria in IspB 53a. a. Coq1 fused with a fragment of
Expression of the -IspB fusion protein restored respiratory defects in the COQ1 deficient strain. Moreover, in this strain, S. Ubiquinone-8 retained by Escherichia coli was synthesized instead of ubiquinone-6 retained by cerevisiae. This result is the first result of successful synthesis of ubiquinone of different species in other organisms, and shows that the side chain of ubiquinone is determined by prenyltransferase. Further, it was revealed that ubiquinone-8 functionally complements ubiquinone-6 in Saccharomyces cerevisiae.

(7) Schizosaccharomy
From ces pombe (S. pombe), the gene encoding decaprenyl diphosphate synthase, which is the side chain of ubiquinone-10, was isolated and cloned by the following strategy.

That is, since there is a conserved region in prenyl diphosphate synthase, a primer for PCR was designed based on these sequences, and S. pomb
PCR was performed using the genome of e as a template. When the amplified fragment was confirmed, a fragment suspected of encoding prenyltransferase could be isolated. Therefore, the entire region was isolated from the genomic and cDNA libraries using the fragment as a probe.

(8) S. prenyl diphosphate synthase isolated from Pombe and S. S. cerevisiae hexaprenyl diphosphate synthase, Bacillus heptaprenyl diphosphate synthase, and E. coli octaprenyl diphosphate synthase. cerevi
siae is N-terminal side and region (I) compared to others
It can be seen that there is a long distance between and area (II). S. pombe
Has 377 amino acids and has a homology of 39.9% with hexaprenyl diphosphate synthase, 30.9% with heptaprenyl diphosphate synthase and 34.0% with octaprenyl diphosphate synthase. Is shown (FIG. 5).

(9) As described above, the S. cerev
Since it was confirmed that 53 amino acids on the N-terminal side of hexaprenyl diphosphate synthase were sufficient for migration to mitochondria in S. isiae, S. Three plasmids were constructed by adding 53 amino acids on the N-terminal side of hexaprenyl diphosphate synthase to prenyl diphosphate synthase isolated from pombe. The first is 53 amino acids with the entire region added, 2
The third is 53 amino acids with 20 amino acids on the N-terminal side deleted, and the third is 53 amino acids with 3 N-terminals.
Constructed by adding 0 amino acids deleted. The three constructed plasmids were transformed into S. It was introduced into a hexaprenyl diphosphate synthase deficient strain of S. cerevisiae (Fig. 6).

As a result of measuring the activity of prenyl diphosphate synthase using these plasmids, the following was revealed (FIG. 7). lane 1 is S. cerev
A wild strain of isiae, lane 2, is S. cerevi
siae hexaprenyl diphosphate synthase deficient strain, l
Ane 3 is S. cerevisiae at the N-terminal 53 amino acids of hexaprenyl diphosphate synthase in the hexaprenyl diphosphate synthase-deficient strain. It is a product in which 20 amino acids at the N-terminal side of prenyl diphosphate synthase isolated from pombe were deleted. As can be seen from lane 3, the decaprenyl diphosphate synthetic activity was confirmed. From this, S. p
It was confirmed that the prenyl diphosphate synthase isolated from ombe was decaprenyl diphosphate synthase. However, S. Respiratory defects could not be complemented due to the deficiency of hexaprenyl diphosphate synthase in S. cerevisiae. This is the S. It is considered that the activity is low in C. cerevisiae or COQ2 cannot recognize decaprenyl diphosphate. Also, the remaining 2 built
No activity could be confirmed for any type of plasmid.

(10) From the above results, the following is clarified. A primer was designed based on the region conserved in prenyl diphosphate synthase, and S. When PCR was carried out using the pombe genome as a template, a fragment suspected of encoding prenyl diphosphate synthase could be isolated. The fragment was used as a probe to isolate the entire region from genomic and cDNA libraries. Results of base sequence determination (SEQ ID NO: 1 in the sequence listing shown in Tables 1 and 2 below)
The entire region conserved in prenyl diphosphate synthase was present.

[0024]

[Table 1]

[0025]

[Table 2]

The isolated prenyl diphosphate synthase was transformed into S.
Transfer of hexaprenyl diphosphate synthase from S. cerevisiae to mitochondria When a signal was introduced into a strain deficient in hexaprenyl diphosphate synthase, the activity of decaprenyl diphosphate synthase was confirmed.

[0027]

The present invention succeeded in expressing the Escherichia coli-derived ispB gene in Saccharomyces cerevisiae by utilizing the transfer sequence of the budding yeast-derived COQ1 gene to mitochondria, and other than ubiquinone-6 originally held by yeast. For the first time, it was possible to generate a heterogeneous ubiquinone, ubiquinone-8. Further, by utilizing the above-mentioned transition sequence, it was possible for the first time to generate the side chain of ubiquinone-10 in the same yeast, and it became possible to freely change the side chain of ubiquinone by genetic engineering.

[Brief description of the drawings]

FIG. 1 shows an HPLC chromatogram showing the construction of plasmids pYE6 and pYE7 and the production of ubiquinone in the strain into which the plasmids were introduced.

FIG. 2 shows S. pneumoniae introduced with pYE6 or pCOQ1. cer
H of ubiquinone extracted from Evisiae YKK6
A PLC chromatogram is shown.

FIG. 3 S. cerevisiae YKK6 / pYE
The thin-layer chromatogram of 6 products is shown.

FIG. 4 shows the structures of pYE6 and pYE7.

FIG. 5: S. Pombe, S. cerevisiae,
B. subtilis, E .; It is a comparison figure of the amino acid sequence of each prenyl transferase of E. coli.

FIG. 6: S. 3 shows three types of plasmids in which the signal sequence-containing region of the hexaprenyl diphosphate synthase gene (COQ1) is added to the prenyl diphosphate synthase gene (DPS) isolated from pombe.

FIG. 7: S. cerevisiae wild strain (wild
type), S.I. cerevisiae hexaprenyl diphosphate synthase gene-deficient strain (YKK6 (ΔCOQ
1)), S. cerevisiae in the 53 amino acids on the N-terminal side of COQ1. pom
FIG. 3 is a diagram showing the activity measurement of decaprenyl diphosphate synthase in a strain (YKK6 / pDPS) to which the N-terminal 20 amino acids of the decaprenyl diphosphate synthase gene isolated from be was added.

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Claims (9)

[Claims] 1. Saccharomyces cere
A heterologous prenyltransferase characterized by fusing a fragment containing a translocation sequence of the structural gene COQ1 of hexaprenyl diphosphate synthase of Visaeae to mitochondria with a heterologous prenyltransferase gene and expressing the resulting fusion gene. How to generate. 2. The method according to claim 1, wherein the translocation sequence is contained within a region encoding the N-terminal 53 amino acids of COQ1. 3. The heterologous prenyl transferase gene is isp derived from Escherichia coli.
B gene or Schizosaccharomyces
It is a decaprenyl diphosphate synthase gene derived from pombe, The method of Claim 1 or Claim 2 characterized by the above-mentioned. 4. The octaprenyl diphosphate synthase is produced by using the ispB gene, and the decaprenyl diphosphate synthase is produced by using the decaprenyl diphosphate synthase gene. The method according to 3. 5. An expression plasmid pYE6 obtained by inserting a fusion gene of a region Coq1 encoding the N-terminal 53 amino acids of COQ1 and an ispB gene into a shuttle vector YEp13M4. 6. The expression plasmid pYE according to claim 5.
6 for Saccharomyces cerevisia
The method for producing ubiquinone-8 is characterized in that it is expressed in e. 7. The expression plasmid pYE according to claim 5.
6 for Saccharomyces cerevisia
The method for producing ubiquinone-6 and ubiquinone-8 is characterized in that the ubiquinone-6 and the ubiquinone-8 are expressed. 8. A primer for PCR is designed based on the region conserved in prenyl diphosphate synthase, and S
PCR was performed using the genome of chizosaccharomyces pombe as a template, the amplified DNA fragment was confirmed, the fragment expected to encode prenyltransferase was isolated, and the fragment was used as a probe to isolate the entire region from the genome and cDNA library. A method for producing a decaprenyl diphosphate synthase gene, which comprises isolating. 9. A decaprenyl diphosphate synthase gene having the sequence represented by SEQ ID NO: 1 in the sequence listing.