Geranyl diphosphate synthase genes

Details Geranyl diphosphate synthase genes Geranyl diphosphate synthase genes

1999-08-16

2002-05-28

Saidha, Tekchand (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S041000, C435S252300, C435S320100, C435S832000, C536S023200

Reexamination Certificate

active

06395525

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a geranyl diphosphate synthase, a gene encoding the synthase, a recombinant vector comprising the gene, and methods for preparing a geranyl diphosphate synthase and geranyl diphosphate, respectively.
BACKGROUND ART
Among those substances which have an important function in organisms, there are a large number of substances biosynthesized with isoprene (2-methyl-1,3-butadiene) units. These compounds are also called isoprenoids, terpenoids or terpenes. Depending on the number of carbon atoms they have, they are classified into hemiterpene (C5), monoterpene (C10), sesquiterpene (C15), diterpene (C20), sesterterpene (C25), triterpene (C30), tetraterpene (C40) and the like.
Actual biosynthesis of these substances starts with the synthesis of isopentenyl diphosphate (IPP), the active isoprene unit. Ultimately, the actual form of the isoprene unit which had been proposed as an putative precursor substance is IPP, the so-called active isoprene unit.
It is known that dimethylallyl diphosphate (DMAPP), an isomer of IPP, is synthesized into an active isoprenoid such as geranyl diphosphate (GPP), neryl diphosphate, farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), geranylfarnesyl diphosphate (GFPP), hexaprenyl diphosphate (HexPP) or heptaprenyl diphosphate (HepPP), through condensation with IPP.
Through cis-condensation of FPP, GPP and the like in which the all-E type is considered to be the active type, a number of compounds such as natural rubber, dolichol, bactoprenol (undecaprenol) or various polyprenols found in plants are synthesized. It is considered that these compounds are synthesized by the consecutive condensation using the energy of phosphate bonds between the pyrophosphoric acid and the carbon skeleton in their molecules. It is considered that pyrophosphoric acid is generated as a by-product of the condensation.
Active type isoprenoid synthases which condensate IPP into allylic substrates of DMAPP, GPP, FPP, GGPP, GFPP, etc. in succession are called prenyl diphosphate synthases or prenyltransferases. Prenyl diphosphate synthases have different designations depending on the number of carbon atoms in their major reaction product. For example, the enzyme which catalyzes the production of farnesyl diphosphate with 15 carbon atoms is called farnesyl diphosphate synthase (FPP synthase); and the enzyme which catalyzes the production of geranylgeranyl diphosphate with 20 carbon atoms is called geranylgeranyl diphosphate synthase (GGPP synthase).
Various prenyl diphosphate synthase genes have already been obtained from bacteria, archaea, fungi, plants and animals. Purification, activity determination as well as gene cloning and DNA sequencing have been reported on FPP synthases, GGPP synthases, hexaprenyl diphosphate synthases, heptaprenyl diphosphate synthases, octaprenyl diphosphate synthases, nonaprenyl diphosphate synthases (solanesyl diphosphate synthases), undecaprenyl diphosphate synthases and the like.
These prenyl diphosphate synthases that are fundamental for the synthesis of important and diversified compounds from both industrial and life-scientific viewpoints are generally unstable and low in specific activity. Thus, industrial application of them could not be expected. In recent several years, however, thermostable FPP synthase genes and GGPP synthase genes have been isolated from thermophilic bacteria and archaea [A. Chen and D. Poulter (1993), J. Biol. Chem., 268 (15), 11002-11007; T. Koyama et al., (1993), J. Biochem. (Tokyo), 113 (3), 355-363; S.-i. Ohnuma et al., (1994), J. Biol. Chem., 269 (20), 14792-14797]. Thus, conditions for utilizing prenyl diphosphate synthases are now being prepared.
Enzymes which synthesize C
10-25
prenyl diphosphates are homodimers. It is relatively easy to allow them to react in vitro, and a number of reports have been made on their reaction. In those enzymes, an enzyme having activity to synthesize GPP (a C
10
prenyl diphosphate) specifically has not been isolated, though partial purification of it has been reported (L. Heide and U. Berger, 1989, Arch, Biochem. Biophys., 273 (2) 331-8). Although it has been reported that a GPP synthase was successfully purified from pig liver (J. K. Dorsey et al., 1966, J. Biol. Chem. 241 (22), 5353-5360), this enzyme catalyzes the synthesis of FPP at the same time. Thus, based on the current definition of prenyl diphosphate synthase, this enzyme should be called FPP synthase.
GPP is the first intermediate for the synthesis of many monoterpenes known and is the most important compound in the biosynthesis pathway of monoterpenes.
Both geraniol and its isomer nerol, which are representative monoterpenes, are aromatics in the major components of rose oil. Another representative monoterpene camphor which is an extract from
Cinnamomum camphora
is also used as a mothball.
However, GPP synthase gene has not been isolated yet.
Under circumstances, a technology is demanded which artificially modifies the amino acid sequence of a thermophile-derived, stable, homodimer type prenyl diphosphate synthase having a high specific activity to thereby engineer a homodimer type, thermostable prenyl diphosphate synthase which specifically catalyzes the synthesis of GPP.
As thermophile-derived prenyl diphosphate synthases,
Bacillus stearothermophilus
FPP synthase and
Sulfolobus acidocaldarius
GGPP synthase have been modified. Mutants of the
S. acidocaldarius
GGPP synthase and genes thereof were selected using as an indicator an ability to complement the glycerol metabolism ability of a HexPP synthesis-dificient
Saccharomyces serevisiae
(budding yeast) [S.-i. Ohnuma et al., (1996), J. Biol. Chem., 271 (31), 18831-18837]. Mutants of the
S. stearothermophilus
FPP synthase having GGPP synthesis activity and genes thereof were obtained using lycopene synthesis as an indicator [S.-i. Ohnuma et al., (1996), J. Biol. Chem., 271 (17), 10087-10095]. Further, 18 mutant enzymes which synthesize a number of prenyl diphosphates from GGPP to HexPP in various proportions, and genes encoding those enzymes were obtained by site-directed mutagenesis the nucleotides encoding the amino acid residue located 5 amino acid residues upstream of the Asp-rich domain conserved region I (DDXX(XX)D) [S.-i. Ohnuma et al., (1996), J. Biol. Chem., 271 (48), 30748-30754]. It has been found that the amino acid residue located 5 amino acid residues upstream of the Asp-rich domain conserved region I (DDXX(XX)D) is involved in the regulation of chain lengths of reaction products.
However, no mutant enzyme having activity to synthesize GPP specifically has been obtained yet.
DISCLOSURE OF THE INVENTION
It is an object of the invention to provide a geranyl diphosphate synthase and a gene encoding the synthase.
As a result of extensive and intensive researches toward the solution of the above problem, the present inventor has succeeded in isolating a geranyl diphosphate synthase and a gene encoding the synthase by replacing a part of the amino acid sequence of a farnesyl diphosphate synthase. Thus, the present invention has been achieved.
The present invention relates to the following recombinant protein (a) or (b):
(a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1;
(b) a protein which consists of the amino acid sequence shown in SEQ ID NO: 1 having deletion, substitution or addition of at least one amino acid excluding the amino acid at position 82, and which has geranyl diphosphate synthase activity.
Further, the present invention relates to a gene coding for the above-described recombinant protein (a) or (b).
Further, the present invention relates to a geranyl diphosphate synthase gene comprising the nucleotide sequence shown in SEQ ID NO: 2.
Further, the present invention relates to a recombinant vector comprising any of the above-described genes.
Further, the present invention relates to a transformant transformed with the above-described recombinant vector.
Further, the present invention relates to a method of preparing a

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