Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
2000-10-10
2002-10-08
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S041000, C435S069100, C435S252330, C435S320100, C536S023200, C536S023600
Reexamination Certificate
active
06461842
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a process for producing coenzyme Q
10
, which can be used as a pharmaceutical agent and the like. In more detail, the present invention relates to a process for producing coenzyme Q
10
by isolating a gene coding for an enzyme responsible for biosynthesizing coenzyme Q
10
side chain, the key enzyme in the coenzyme Q
10
biosynthetic pathway, i.e. decaprenyl diphosphate synthase, from a bacterium belonging to the family Rhizobiaceae, and transferring said gene into a microorganism to produce coenzyme Q
10
.
2. Description of the Related Art
Industrially, coenzyme Q
10
has been produced by isolating a coenzyme from plants such as tobacco and synthetically altering the side chain.
In addition, it has been known that coenzyme Q
10
is produced by a wide variety of organisms including from microorganisms such as bacterium and yeast, to higher plants and animals. One of the most effective processes for producing coenzyme Q
10
is believed to be a process which comprises culturing the microorganism and extracting the compound from the culture. Said process has also been used in industrial production of coenzyme Q
10
. However, the above known processes do not provide enough productivity because of their low yield or complicated operation.
Though there are some differences between the prokaryotic and eucaryotic pathways for biosynthesis of coenzyme Q
10
, both pathways consist of complicated multi-step reactions in which many enzymes are involved. They basically consist of the following three steps; synthesizing decaprenyl diphosphate, which is used for the prenyl side chain of coenzyme Q
10
; synthesizing parahydroxy benzoic acid, which is used for the quinone ring; and binding those two components and converting the substituents sequentially to give coenzyme Q
10
. The reaction, which determines the length of the side chain and is believed to be the rate-limiting step in the biosynthetic pathway, that is, the reaction in which decaprenyl diphosphate synthase is involved, is believed to be the most important reaction. Therefore, in order to produce coenzyme Q
10
effectively, it might be a good idea to isolate the decaprenyl diphosphate synthase gene, the key gene in the biosynthetic pathway, and to use said gene for improving productivity. One of the potential candidates for the gene sources is a bacterium belonging to the family Rhizobiaceae, which produces relatively large amounts of coenzyme Q
10
.
Until now, decaprenyl phosphoric acid synthase genes had been isolated from several microorganisms including
Schizosaccaromyces pombe
(Japanese Patent Application Laid Open No. H09-173076) and
Gluconobacter suboxydans
(Japanese Patent Application Laid Open Open No. H10-57072). However, they do not show enough productivity for coenzyme Q
10
and therefore, the art had not yet achieved effective culture, isolation or purification with those microorganisms. Accordingly, it has been desired to isolate a gene of said enzyme from a microorganism having high coenzyme Q
10
producing ability.
SUMMARY OF THE INVENTION
The present invention has been made to solve the above-mentioned problem of low productivity. The object of the present invention is to provide a process for producing coenzyme Q
10
effectively by means of a microorganism, by isolating a gene coding for coenzyme Q
10
-side chain synthetic enzyme from a bacterium belonging to the family Rhizobiaceae and using the gene.
According to the present invention, decaprenyl diphosphate synthase gene, the key gene in the biosynthetic pathway for coenzyme Q
10
, was isolated from a bacterium belonging to the family Rhizobiaceae. An effective coenzyme Q
10
production has been achieved by transferring the gene into a microorganism such as
Escherichia
-
coli
and expressing therein.
The inventors have tried to isolate the gene of decaprenyl diphosphate synthase from a bacterium belonging to the family Rhizobiaceae which produces relatively large amounts of coenzyme Q
10
and succeeded in isolating said gene.
Accordingly, the present invention provide a DNA comprising a DNA sequence of the Seq. ID No. 1, or a sequence having deletion, addition or insertion of one or more base in the sequence and coding for decaprenyl diphosphate synthase. The present invention also provide a protein having an amino acid sequence of the Seq. ID No. 2, or an amino acid sequence having deletion, addition or insertion of one or more amino acid in said sequence and having decaprenyl diphosphate synthase activity; and a DNA encoding said amino acid sequence.
The present invention also provide a process for producing coenzyme Q
10
comprising the steps of transferring the above described DNA sequence into a host microorganism and culturing the microorganism. The host microorganism used in the present invention is not limited but preferably is
Escherichia coli.
Although a normal
Escherichia coli
produces coenzyme Q
8
, according to the present invention,
Escherichia coli
can be modified to produce coenzyme Q
10
.
In addition, the present invention provides an expression vector comprising the above-described DNA sequence. The expression vector of the present invention may be constructed by using any of known vector systems. For example, pQAD-1, which is constructed by transferring the gene of the Seq. ID No. 1 into the known expression vector system pUCNT, is provided.
According to the invention, a host microorganism transformed with the above-described DNA sequence is also provided. For the host microorganism in the present invention,
Escherichia coli
is preferably used.
REFERENCES:
patent: 6103488 (2000-08-01), Matsuda et al.
patent: 10-057072 (1998-03-01), None
patent: 11-056372 (1999-03-01), None
patent: 11-178590 (1999-07-01), None
H. Matsuda et al., “Molecular colning and mutational analysis of the ddsA gene encoding decaprenyl disphosphate synthase from Gluconobacter suboxydans”, Eur. J. Biochem., (1998), vol. 225, No. 1, pp. 52-59.
M. Kawamukai et al., “Analysis of the decaprenyl disphosphate synthase (dps) gene in fission yeast suggests a role of ubiquinone as an antioxidant”, J. Biochem., (1997), vol. 121, No. 3, pp. 496-505.
Kawamukai M. Accession AB006850. Aug. 11, 1998 (Alignment No. 1).*
Kawamukai M. Accession AB006850. Aug. 11, 1998 (Alignment No. 2).*
Okada et al. Molecular cloning and mutational analysis of the ddsA gene encoding decaprenyl disphosphate synthase from Gluconobacter suboxydans. Eur J Biochem Jul. 1, 1998;255(1):52-9.
Hasegawa Junzo
Ikenaka Yasuhiro
Kawamukai Makoto
Matsuda Hideyuki
Nishi Kenichi
Achutamurthy Ponnathapu
Fronda Christian L.
Kaneka Corporation
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