Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
2001-07-23
2004-07-13
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
C435S041000, C435S193000, C435S252300, C435S252330, C435S254110, C435S320100, C536S023200
Reexamination Certificate
active
06762037
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a process for producing a coenzyme Q
10
for pharmaceutical and other uses. More particularly, the invention relates to a process for producing coenzyme Q
10
which comprises isolating a gene coding for the coenzyme Q
10
side-chain synthase, which is a key enzyme involved in the biosynthesis of coenzyme Q
10
, i.e. decaprenyl diphosphate synthase, from a fungal strain of the genus Saitoella and introducing it into a host microorganism to let it elaborate coenzyme Q
10
.
BACKGROUND ART
The conventional technology for commercial production of coenzyme Q
10
comprises isolating the coenzyme from a tobacco or other plant and modifying the length of its side chain by a synthetic technique.
While it is known that coenzyme Q
10
is produced by a broad spectrum of organisms ranging from microorganisms, such as bacteria and yeasts, to higher animals and plants, the method comprising culturing a microorganism and extracting coenzyme Q
10
from the microorganism is regarded as one of the most effective production methods and has actually been exploited commercially. However, the prior art methods are invariably poor in productivity, providing for only low outputs and/or involving time-consuming procedures.
The pathways for biosynthesis of coenzyme Q
10
in organisms are partly different between the prokaryote and the eukaryote but invariably comprise a complicated cascade of reactions involving many kinds of enzymes. However, these pathways are basically comprised of three fundamental steps, namely the step of synthesizing decaprenyl diphosphate as the precursor of the prenyl side-chain of coenzyme Q
10
, the step of synthesizing p-hydroxybenzoic acid as the basis of the quinone ring of coenzyme Q
10
, and the step of coupling these two compounds together and effecting a serial substituent transformation to complete coenzyme Q
10
. Of these reactions, the reaction determinant of the length of the side-chain of coenzyme Q
10
and acknowledged to be the rate-determining step of its biosynthesis, i.e. the reaction catalyzed by decaprenyl diphosphate synthase, is considered to be the most important reaction. Therefore, in order that coenzyme Q
10
may be produced with good efficiency, it seems worthwhile to isolate the key gene involved in said biosynthesis, namely the gene coding for decaprenyl diphosphate synthase, and utilize it for enhanced production of the enzyme. As sources of the gene, fungi capable of producing coenzyme Q
10
in comparatively large amounts can be regarded as useful candidates.
Heretofore, genes coding for decaprenyl diphosphate synthase have been isolated from several kinds of microorganisms, such as
Schizosacch aromyces pombe
(JP09-173076A) and
Gluconobacter suboxydans
(JP10-57072A), etc., but the inherent coenzyme Q
10
productivity of these micrcorganisms cannot be considered high enough and neither an efficient cultural protocol for these microorganisms nor an efficient isolation and purification procedure has been established as yet. Therefore, there has been a standing demand for isolation of a coenzyme Q
10
-encoding gene from a microorganism capable of highly producing a coenzyme Q
10
.
Devoted to providing a solution to the above-mentioned production problems, the present invention has for its object to isolate a gene coding for the enzyme synthesizing the coenzyme Q
10
side chain from a fungal strain of the genus Saitoella and exploit it to advantage for the efficient microbial production of coenzyme Q
10
.
DISCLOSURE OF THE INVENTION
To accomplish the above object, in the present invention, the key gene involved in the biosynthesis of coenzyme Q
10
, namely the gene coding for decaprenyl diphosphate synthase, was isolated from a fungal strain of the genus Saitoella in the first place. Then, this gene was introduced and allowed to be expressed in a host microorganism, such as
Escherichia coli
, to thereby enable the host to produce coenzyme Q
10
with efficiency.
The inventors of the present invention made intensive investigations for isolating such genes coding for decaprenyl diphosphate synthase from fungal strains of the genus Saitoella capable of producing comparatively large amounts of coenzyme Q
10
and have succeeded in isolating said genes.
The present invention, therefore, is concerned with a DNA of the following (a), (b) or (c).
(a) a DNA having the nucleotide sequence shown under SEQ ID NO:1
(b) a DNA having a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO:1 by the deletion, addition, insertion and/or substitution of one or a plurality of nucleotides
and coding for a protein having decaprenyl diphosphate synthase activity
(c) a DNA which hybridizes with the DNA having the nucleotide sequence of SEQ ID NO:1 under a stringent condition and codes for a protein having decaprenyl diphosphate synthase activity.
The present invention is further concerned with a protein of the following (d) or (e).
(d) a protein having the amino acid sequence shown under SEQ ID NO:2
(e) a protein having an amino acid sequence derived from the amino acid sequence of SEQ ID NO:2 by the deletion, addition, insertion and/or substitution of one or a plurality of amino acids
and having decaprenyl diphosphate synthase activity.
The invention is further concerned with a DNA coding for this protein.
The present invention is further concerned with an expression vector containing said DNA. For the expression vector of the invention, various vector systems heretofore known can be utilized and, therefore, may for example be pNTSal as constructed by cloning the DNA having the sequence of SEQ ID NO:1 into the vector pUCNT for expression.
The present invention is further concerned with a transformant as constructed by transforming a host microorganism with said DNA. As the host microorganism for the invention,
Escherichia coli
can be used with advantage.
The invention is further concerned with a process for producing coenzyme Q
10
which comprises culturing said transformant in a culture broth and harvesting the coenzyme Q
10
produced and accumulated in the resulting culture. The host microorganism for this process is not particularly restricted but may be
Escherichia coli
to mention a preferred example. The coenzyme Q produced by
Escherichia coli
is coenzyme Q
8
but the invention enables this microorganism to produce coenzyme Q
10
.
The inventors made intensive investigations on the isolation of the enzyme gene from a fungal strain which belongs to the genus Saitoella and is capable of producing comparatively large amounts of coenzyme Q
10
and succeeded in acquiring a fragment of the particular gene by a PCR technique.
The inventors compared the sequence of the known gene coding for decaprenyl diphosphate synthase with the genes cording for polyprenyl diphosphate synthases, namely long-chain prenyl synthases which are analogous to said known enzyme gene but differ from the same in chain length and, for the region of high homology, synthesized various PCR primers. Using these primers in various combinations, they studied PCR conditions. As a result, they found by analysis of the gene sequence that when a PCR using DPS-1 (SEQ ID NO:3) and DPS-1 1AS (SEQ ID NO:4) as primers is carried out according to the protocol of heat-treatment at 94° C.×3 minutes, followed by 40 cycles of 94° C., 1 minute→43° C., 2 min→72° C., 2 minutes, a ca 220 bp fragment of the enzyme gene can be amplified from the chromosome gene of
Saitoella complicata
IFO 10748, a fungus belonging to the genus Saitoella.
Then, to acquire the full length of this enzyme gene, the chromosome gene
Saitoella complicata
IFO 10748 is digested with the restriction enzyme EcoRI and inserted into a &lgr; phage vector to construct a recombinant phage library. After the plaque is transferred to a nylon membrane, the plaque hybridization is carried out using the labeled PCR fragment, whereby a clone having the full-length decaprenyl diphosphate synthase gene can be obtained.
Sequencing of the decaprenyl diphosphate synthase gene oc
Hasegawa Junzo
Ikenaka Yasuhiro
Kawamukai Makoto
Matsuda Hideyuki
Takahashi Satomi
Connolly Bove & Lodge & Hutz LLP
Kaneka Corporation
Slobodyansky Elizabeth
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