U.S. patent number RE43,425 [Application Number 09/902,651] was granted by the patent office on 2012-05-29 for mutant prenyl diphosphate synthase.
This patent grant is currently assigned to Toyota Jidosha Kabushiki Kaisha. Invention is credited to Kazutake Hirooka, Hiroyuki Nakane, Tokuzo Nishino, Shinichi Ohnuma, Chikara Ohto.
United States Patent |
RE43,425 |
Nakane , et al. |
May 29, 2012 |
Mutant prenyl diphosphate synthase
Abstract
A mutant prenyl diphosphate synthase capable of synthesizing
prenyl diphosphates, shorter than those synthesized by the original
enzyme, by modifying the amino acid sequence in and upstream of the
aspartic acid-rich domain DDXX (XX)D (X denotes any amino acid, and
XX in the parentheses may not be present) present in region II of
the prenyl diphosphate synthase.
Inventors: |
Nakane; Hiroyuki (Toyota,
JP), Ohto; Chikara (Toyota, JP), Ohnuma;
Shinichi (Sendai, JP), Hirooka; Kazutake (Sendai,
JP), Nishino; Tokuzo (Sendai, JP) |
Assignee: |
Toyota Jidosha Kabushiki Kaisha
(Toyota-shi, JP)
|
Family
ID: |
16635389 |
Appl.
No.: |
09/902,651 |
Filed: |
July 12, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
08898560 |
Jul 22, 1997 |
5935832 |
Aug 10, 1999 |
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Foreign Application Priority Data
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Jul 24, 1996 [JP] |
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08-213211 |
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Current U.S.
Class: |
435/193;
536/23.2; 435/410; 435/254.11; 435/252.3; 435/320.1; 435/325 |
Current CPC
Class: |
C12N
9/1085 (20130101) |
Current International
Class: |
C12N
9/10 (20060101); C12N 1/20 (20060101); C07H
21/04 (20060101); C12N 15/00 (20060101) |
Field of
Search: |
;435/193,320.1,325,419,252.3,254.11,132 ;536/23.2,23.7 |
Foreign Patent Documents
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Apr 1993 |
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Sep 1995 |
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0 699 761 |
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Mar 1996 |
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0 733 709 |
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Sep 1996 |
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409065878 |
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JP |
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Primary Examiner: Steadman; David J
Attorney, Agent or Firm: Kenyon & Kenyon LLP
Claims
We claim:
1. A mutant prenyl diphosphate synthase having .[.a modified.].
.Iadd.the .Iaddend.amino acid sequence.[.,.]. .[.wherein said
mutant prenyl diphosphate synthase comprises an aspartic acid-rich
domain having the sequence,
D.sub.1D.sub.2X.sub.1X.sub.2(X.sub.3X.sub.4)D.sub.3, in region II
of said mutant prenyl diphosphate synthase, wherein each of
D.sub.1, D.sub.2 and D.sub.3 denote an aspartic acid residue;
X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are each independently any
amino acid and X.sub.3 and X.sub.4 are each optionally
independently present in the aspartic acid rich domain, and wherein
said mutant prenyl diphosphate synthase comprises (1) at least one
amino acid substitution, said at least one amino acid substitution
located at at least one ammo acid position selected from (a) an
amino acid between D.sub.1 and the amino acid residue at the fifth
position upstream of D.sub.1 and (b) the amino acid residue located
one amino acid position upstream of D.sub.3; (2) at least one
additional amino acid inserted between D.sub.3 and the first amino
acid upstream of D.sub.3; or a combination of (2) and (3); wherein
said mutant prenyl diphosphate synthase synthesizes prenyl
diphosphate which is shorter than prenyl diphosphate synthesized by
a corresponding wild-type enzyme.]. .Iadd.of SEQ ID NO:1, except
that: threonine at position 78 of SEQ ID NO:1 is replaced with
phenylalanine and histidine at position 81 of SEQ ID NO:1 is
replaced with alanine; threonine at position 78 of SEQ ID NO:1 is
replaced with phenylalanine and histidine at position 81 of SEQ ID
NO:1 is replaced with leucine; phenylalanine at position 77 of SEQ
ID NO:1 is replaced with tyrosine, threonine at position 78 of SEQ
ID NO:1 is replaced with phenylalanine, and histidine at position
81 of SEQ ID NO:1 is replaced with leucine; phenylalanine at
position 77 of SEQ ID NO:1 is replaced with tyrosine, threonine at
position 78 of SEQ ID NO:1 is replaced with phenylalanine, and
histidine at position 81 of SEQ ID NO:1 is replaced with alanine;
or phenylalanine at position 77 of SEQ ID NO:1 is replaced with
tyrosine, threonine at position 78 of SEQ ID NO:1 is replaced with
serine, valine at position 80 of SEQ ID NO:1 is replaced with
isoleucine, isoleucine at position 84 of SEQ ID NO:1 is replaced
with leucine, and proline and serine are inserted sequentially
between position 84 and position 85 of SEQ ID NO:1.Iaddend..
.[.2. A mutant prenyl diphosphate synthase according to claim 1
wherein said mutant has the enzymatic activities and thermo
stability of wild type prenyl diphosphate synthase..].
3. A mutant .[.enzyme.]. .Iadd.prenyl diphosphate synthase
.Iaddend.according to claim 1 wherein .[.the.]. .Iadd.a
.Iaddend.reaction product of the .Iadd.mutant .Iaddend.prenyl
diphosphate synthase is farnesyl diphosphate.
4. A mutant .[.enzyme.]. .Iadd.prenyl diphosphate synthase
.Iaddend.according to claim 1 wherein the .Iadd.mutant
.Iaddend.prenyl diphosphate synthase .[.is of the homodimer-type.].
.Iadd.forms a homodimer.Iaddend..
.[.5. A mutant enzyme according to claim 1 wherein the prenyl
diphosphate synthase is derived from archaea..].
6. A mutant .[.enzyme.]. .Iadd.prenyl diphosphate synthase
.Iaddend.according to claim 1 wherein the .Iadd.mutant
.Iaddend.prenyl diphosphate synthase is .[.derived from.]. .Iadd.a
mutant of a .Iaddend.Sulfolobus acidocaldarius .Iadd.prenyl
diphosphate synthase.Iaddend..
7. A mutant .[.enzyme.]. .Iadd.prenyl diphosphate synthase
.Iaddend.according to claim 1 wherein the .Iadd.mutant
.Iaddend.prenyl diphosphate synthase is .[.a.]. .Iadd.more
.Iaddend.thermostable .[.enzyme.]. .Iadd.at 70.degree. C. or
80.degree. C. than the wild-type geranylgeranyl diphosphate
synthase of Sulfolobus acidocaldarius.Iaddend..
.[.8. A mutant prenyl diphosphate synthase according to claim 1,
wherein at least one amino acid selected from phenylalanine at
position 77, threonine at position 78, valine at position 80,
histidine at position 81, and isoleucine at position 84 has been
substituted by another amino acid, or one or more amino acids have
been inserted in between isoleucine at position 84 and methionine
at position 85 in the geranylgeranyl diphosphate synthase as set
forth in SEQ ID No: 1..].
.[.9. A mutant prenyl diphosphate synthase according to claim 1
wherein at least one amino acid selected from phenylalanine at
position 77, threonine at position 78, valine at position 80,
histidine at position 81, and isoleucine at position 84 has been
substituted by another amino acid, and/or two amino acids have been
inserted in between isoleucine at position 84 and methionine at
position 85 in the geranylgeranyl diphosphate synthase as set forth
in SEQ ID NO: 1, wherein the phenyl alanine at position 77 has been
replaced with tyrosine, the threonine at position 78 has been
replaced with phenylalanine or serine, the valine at position 80
has been replaced with isoleucine, the histidine at position 81 has
been replaced with leucine or alanine, or the isoleucine at
position 84 has been replaced with leucine; or proline and serine
have been inserted in between the isoleucine at position 84 and the
methionine at position 85..].
.[.10. A mutant prenyl diphosphate synthase according to claim 1,
wherein the mutant prenyl diphosphate synthase is derived from a
native geranylgeranyl diphosphate synthase of an organism selected
from the group consisting of Arabidopsis thaliana, Lupinas albus,
Capsicum annuum, Sulfolobus acidocaldarius, Rhodobactor
sphaeroides, Rhodobactor capsulatus, Erwinia herbicola, Myxococcus
thaliana and Neurospora crassa..].
11. A DNA encoding .[.an enzyme.]. .Iadd.the mutant prenyl
diphosphate synthase .Iaddend.according to claim 1.
12. An RNA .[.transcribed from a DNA according to claim 11.].
.Iadd.encoding the mutant prenyl diphosphate synthase according to
claim 1.Iaddend..
13. A recombinant vector comprising .[.a.]. .Iadd.the .Iaddend.DNA
according to claim 11.
14. .[.A.]. .Iadd.An isolated .Iaddend.host .[.organism.].
.Iadd.cell .Iaddend.transformed with .[.a.]. .Iadd.the
.Iaddend.recombinant vector according to claim 13.
15. A process for producing a mutant .[.enzyme.]. .Iadd.prenyl
diphosphate synthase .Iaddend.according to claim 1, said method
comprising the steps of culturing .[.a.]. .Iadd.an isolated
.Iaddend.host .Iadd.cell .Iaddend.transformed with an expression
vector comprising a DNA .[.coding for.]. .Iadd.encoding
.Iaddend.the mutant .[.enzyme.]. .Iadd.prenyl diphosphate synthase
.Iaddend.and .[.of.]. harvesting the .[.expression product.].
.Iadd.mutant prenyl diphosphate synthase according to claim 1
.Iaddend.from the culture.Iadd., wherein the mutant prenyl
diphosphate synthase is produced by expression of the expression
vector.Iaddend..
16. A process for producing a prenyl diphosphate having not more
than 15 carbons comprising the step of bringing .[.an enzyme.].
.Iadd.the mutant prenyl diphosphate synthase .Iaddend.according to
.[.claim.]. .Iadd.any one of claims .Iaddend.1 .[.or any of claims
2 to 10.]. .Iadd.3, 4, 6 and 7 .Iaddend.or .[.an enzyme.].
.Iadd.the mutant prenyl diphosphate synthase .Iaddend.produced by
the method according to claim 15 into contact with a substrate
selected from the group consisting of isopentenyl diphosphate,
dimethylallyl diphosphate, and geranyl diphosphate.
.Iadd.17. The mutant prenyl diphosphate synthase of claim 1 having
the amino acid sequence of SEQ ID NO:1 except that threonine at
position 78 of SEQ ID NO:1 is replaced with phenylalanine and
histidine at position 81 of SEQ ID NO:1 is replaced with
alanine..Iaddend.
.Iadd.18. The mutant prenyl diphosphate synthase of claim 1 having
the amino acid sequence of SEQ ID NO:1 except that threonine at
position 78 of SEQ ID NO:1 is replaced with phenylalanine and
histidine at position 81 of SEQ ID NO:1 is replaced with
leucine..Iaddend.
.Iadd.19. The mutant prenyl diphosphate synthase of claim 1 having
the amino acid sequence of SEQ ID NO:1 except that phenylalanine at
position 77 of SEQ ID NO:1 is replaced with tyrosine, threonine at
position 78 of SEQ ID NO:1 is replaced with phenylalanine, and
histidine at position 81 of SEQ ID NO:1 is replaced with
leucine..Iaddend.
.Iadd.20. The mutant prenyl diphosphate synthase of claim 1 having
the amino acid sequence of SEQ ID NO:1 except that phenylalanine at
position 77 of SEQ ID NO:1 is replaced with tyrosine, threonine at
position 78 of SEQ ID NO:1 is replaced with phenylalanine, and
histidine at position 81 of SEQ ID NO:1 is replaced with
alanine..Iaddend.
.Iadd.21. The mutant prenyl diphosphate synthase of claim 1 having
the amino acid sequence of SEQ ID NO:1 except that phenylalanine at
position 77 of SEQ ID NO:1 is replaced with tyrosine, threonine at
position 78 of SEQ ID NO:1 is replaced with serine, valine at
position 80 of SEQ ID NO:1 is replaced with isoleucine, isoleucine
at position 84 of SEQ ID NO:1 is replaced with leucine, and proline
and serine are inserted sequentially between position 84 and
position 85 of SEQ ID NO:1..Iaddend.
.Iadd.22. The mutant prenyl diphosphate synthase of claim 1,
wherein the mutant synthesizes more farnesyl diphosphate than the
wild-type geranylgeranyl diphosphate synthase of Sulfolobus
acidocaldarius..Iaddend.
Description
BACKGROUND OF INVENTION
1. Field of Invention
The present invention relates to a novel mutant enzyme which
synthesizes linear prenyl diphosphates that are precursors of
compounds, important for organisms, such as steroids, ubiquinones,
dolichols, carotenoids, prenylated proteins, animal hormones, plant
hormones, and the like; a genetic system encoding said enzyme; and
a method for producing and using said enzyme.
2. Related Art
Of the substances having important functions in organisms, many are
biosynthesized using isoprene (2-methyl-1,3-butadiene) as a
constituent .[.units.]. .Iadd.unit.Iaddend.. These compounds are
also called isoprenoids, terpenoids, or terpenes, and are
classified depending on the number of carbon atoms into
hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15),
diterpenes (C20), sesterterpenes (C25), triterpenes (C30),
tetraterpenes (C40), and the like. The actual biosynthesis starts
with the mevalonate pathway through which mevalonic
acid-5-diphosphate is synthesized, followed by the synthesis of
isopentenyl diphosphate (IPP) which is an active isoprene unit.
The identity of the isoprene unit that was proposed as a precursor
was found to he isopentenyl diphosphate, the so-called active
isoprene unit. Dimethylallyl diphosphate (DMAPP), an isomer of
isopentenyl diphosphate, being used as a substrate in the synthesis
of isopentenyl adenine which is known as a cytokinin, one of the
plant hormones, it is also known to undergo a condensation reaction
with isopentenyl diphosphate to synthesize chain-form active
isoprenoids such as geranyl diphosphate (GPP), neryl diphosphate,
farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP),
geranylfarnesyl diphosphate (GFPP), hexaprenyl diphosphate (HexPP),
heptaprenyl diphosphate (HepPP), and the like.
There are Z type and E type condensation reactions. Geranyl
diphosphate is a product of E type condensation and neryl
diphosphate is of Z type condensation. Although, the all-E type is
considered to be the active form in farnesyl diphosphate and
geranylgeranyl diphosphate, the Z type condensation reaction leads
to the synthesis of natural rubber, dolichols, bactoprenols
(undecaprenols), and .[.plants.]. various polyprenols found in
.Iadd.plants.Iaddend.. They are believed to undergo the
condensation reaction using the phosphate ester bond energy of the
pyrophosphate and the carbon backbone present in the molecule and
to produce pyrophosphate as the byproduct of the reaction.
Farnesyl diphosphate or geranylgeranyl diphosphate serve as a
reaction substrate leading to the synthesis of prenylated proteins
(from farnesyl diphosphate or geranylgeranyl diphosphate)
represented by G proteins that are important in the mechanism of
signal transducer in the cell; cell membrane lipids (from
geranylgeranyl diphosphate) of archaea; squalene (from farnesyl
diphosphate) which is a precursor of steroids; and phytoene (from
geranylgeranyl diphosphate) which is a precursor of carotenoids.
Prenyl diphosphates from hexaprenyl diphosphate and heptaprenyl
diphosphate having six and seven isoprene units, respectively, to
prenyl diphosphates having ten isoprene units serve as the
precursors of the synthesis of ubiquinone and menaquinone (vitamin
K2) that work in the electron transport system.
Furthermore, via the biosynthesis of these active-form isoprenoids,
a vast number of kinds of compounds that are vital to life have
been synthesized. Just to mention a few, there are cytokinins that
are plant hormones and isopentenyl adenosine-modified tRNA that use
hemiterpenes as their precursor of synthesis, .[.geraniols.].
.Iadd.geraniol .Iaddend.and .[.that.]. .Iadd.its
.Iaddend.isomer.Iadd., .Iaddend.nerol.Iadd., .Iaddend.belonging to
monoterpens .Iadd.that .Iaddend.are the main components of rose oil
perfume and a camphor tree extract, camphor, which is an
insecticide. Sesquihormones include juvenile hormones of insects,
diterpenes include a plant hormone gibberellin, trail pheromones of
insects, and retinols and retinals that function as the visual
pigment precursors, binding components of the purple membrane
proteins of highly halophilic archaea, and vitamin A.
Furthermore, using squalene, a triterpene, a wide variety of
steroid compounds have been synthesized, including, for example,
animal sex hormones, vitamin D, ecdysone which is an ecdysis
hormone of insects, a plant hormone brassinolide, constitution of
the plasma membrane etc. Various carotenoids of tetraterpenes that
are precursors of various pigments of organisms and vitamin A are
also important compounds derived from active isoprenoids. Compounds
such as chlorophyll, pheophytin, tocopherol (vitamin E), and
phylloquinone (vitamin K1) are also derived from tetraterpenes.
The active isoprenoid synthases that sequentially condense
isopentenyl diphosphates with such allylic substrates as
dimethylallyl diphosphate, geranyl diphosphate, farnesyl
diphosphate, geranylgeranyl diphosphate, geranylfarnesyl
diphosphate, etc. are called the prenyl diphosphate synthases, and
are also called, based on the name of the compound having the
maximum chain length of the major reaction products, for example
farnesyl diphosphate synthase (FPP synthase), geranylgeranyl
diphosphate (GGPP synthase), and the like. There are reports on
purification, activity measurement, genetic cloning, and sequencing
of the DNA encoding enzymes such as farnesyl diphosphate synthase,
geranylgeranyl diphosphate synthase, hexaprenyl diphosphate
synthase, heptaprenyl diphosphate synthase, octaprenyl diphosphate
synthase, nonaprenyl diphosphate synthase (solanesyl diphosphate
synthase), undecaprenyl diphosphate synthase, and the like from
bacteria, archaea, fungi, plants, and animals.
These active isoprenoid synthases constituting the basis of
chemical synthesis of a great variety of compounds that are
important both in the industry and in the academic field of life
sciences have had few practical uses in the industrial application
due to their unstable nature and low specific activities. However,
with the isolation of thermostable prenyl diphosphate synthases
from thermophilic bacteria and archaea and the genes encoding these
enzymes, their availability as the enzyme has increased.
With regard to farnesyl diphosphate synthase, a gene was isolated
from Bacillus stearothermophilus, a medium thermophile, and an
enzyme having a medium thermal stability was prepared using
Escherichia coli as host cell [T. Koyama et al. (1993) J. Biochem.,
113: 355 363; Japanese Unexamined Patent Publication No.
5(1993)-219961]. With regard to geranylgeranyl diphosphate
synthase, a gene was isolated from high thermophiles such as
Sulfolobus acidocaldarius and Thermus thermophiles [S. -i. Ohnuma
et al., (1994) J. Biol. Chem., 269: 14792 14797; Japanese
Unexamined Patent Publication No. 7(1995)-308193, and; Japanese
Unexamined Patent Publication No. 7(1995)-294956], and enzymes
having a high thermal stability were prepared.
Furthermore, with regard to the prenyl diphosphate synthase having
the functions of both of the farnesyl diphosphate synthase and the
geranylgeranyl diphosphate synthase, the enzyme and the gene
encoding it have been isolated from highly thermophile
Methanobacterium thermoautotrophicum [A. Chen and D. Poulter (1993)
J. Biol. Chem., 268: 11002 11007; A. Chen and D. Poulter (1994)
ARCHIVES OF BIOCHEMSTRY AND BIOPHYTSICS 314], and the thermostable
nature of the enzyme has been demonstrated.
However, in the synthesis of farnesyl diphosphate/geranylgeranyl
diphosphate derived from Methanobacterium thermoautotrophicum,
there are no reports on the data of thin layer chromatography
analysis etc. that can specify the chain length of the reaction
products in connection with the assay of the enzymatic activity;
the chain length has been estimated by measuring geranyl
diphosphate as the allylic substrate. Since geranyl diphosphate can
also serve as a substrate of geranylgeranyl diphosphate synthase,
it is unlikely that the measured activity includes that of the
farnesyl diphosphate synthase alone.
Moreover, the presence of farnesyl diphosphate synthase has not
been confirmed in archaea that are expected to have enzymes having
higher thermo stability, higher salt-stability and
lower-pH-stability.
As mentioned above, the use of the farnesyl diphosphate synthase
derived from Bacillus stearothermophilus resolved part of the
problem of the enzyme being unstable and difficult to handle. But,
an enzyme having a higher thermal stability would be more stable
and more amenable to industrial application.
Moreover, some prenyl diphosphate synthases having a longer chain
length use farnesyl diphosphate as a substrate. When such a
long-chain prenyl diphosphate synthase is used simultaneously with
a farnesyl diphosphate synthase for the purpose of providing the
substrate of the former enzyme, the latter enzyme must have
stability which is equal to or higher than that of the long-chain
prenyl diphosphate synthase. When industrial production of farnesyl
diphosphate is contemplated, the enzyme must be immobilized or
recovered for recycling. When it is regenerated, the enzyme itself
to be more stable, must have higher thermo stability, higher salt
stability, and higher stability in a wider range of pH.
It has been found out that of the two aspartic acid-rich domains
that have been proposed based on the amino acid sequence of the
prenyl diphosphate synthase, the amino acid residue located at the
fifth position in the N-terminal direction from the conserved
sequence I (DDXX(XX)D) (wherein X denotes any amino acid, and the
two X's in the parentheses may not be present) of the aspartic
acid-rich domain in the amino-terminal side is responsible for
controlling the chain length of the reaction product. Hence, a
method has been invented that controls the reaction product for the
purpose of lengthening the chain length of the reaction product
[Japanese patent application No. 8-191635 filed on Jul. 3, 1996
under the title of "A Mutant Prenyl Diphosphate Synthase"]. The
enzyme produced using the method enables production of reaction
products that have several chain lengths. However, methods .[.have
not been.]. .Iadd.are .Iaddend.not known that .[.induce mutation.].
.Iadd.include mutations .Iaddend.of geranylgeranyl diphosphate
synthase to control the reaction products .[.to be in the short
chain-length side.]. .Iadd.having a shorter chain length in order
.Iaddend.to produce farnesyl diphosphate.
SUMMARY OF INVENTION
It is an object of the invention to establish a process for
producing farnesyl diphosphate synthases by modifying amino acid
sequences of prenyl diphosphate enzymes. A new enzyme that is more
stable or that has a high specific activity more adaptable to
industrial application would make it possible to obtain immediately
a mutant prenyl diphosphate synthase or the gene thereof that
produces farnesyl diphosphate and that retains the property
.[.owned.]. .Iadd.exhibited .Iaddend.by .[.the.]. the prenyl
diphosphate synthase prior to mutation.
From the information on the nucleotide sequence of the gene of the
geranylgeranyl diphosphate synthase of the mutant Sulfolobus
acidocaldarius (S. acidocaldarius), it was clarified that out of
the two Aspartic acid-rich domains that have been proposed based on
the analysis of the amino acid sequence of prenyl diphosphate
synthases, the amino acid residues within the aspartic acid-rich
domain conserved sequence I (DDXX(XX)D) at the amino terminal side
or the five amino acid residues to the N-terminal side from the
amino terminal of said conserved sequence I are involved in the
control of chain length of the reaction products.
Thus, the present invention provides a mutant prenyl diphosphate
synthase having a modified amino acid sequence, wherein
at least one amino acid residue selected from (a) the amino acid
residues in between the amino acid residue located at the fifth
position in the N-terminal direction from D of the N-terminal and
the amino acid residue located at the first position in the
N-terminal direction from D of said N-terminal of the aspartic
acid-rich domain DDXX(XX)D (wherein X sequence denotes any amino
acid, and the two X's in the parentheses may not be present)
present in region II, and (b) the amino acid residue located at the
position in the N-terminal direction from D of the C-terminal of
said aspartic acid-rich domain has been substituted by another
amino acid, and/or
additional amino acid(s) have been inserted in between the amino
acid residue located at the first position in the N-terminal
direction from D of the C-terminal and D of said C-terminal of said
aspartic acid-rich domain.
The present invention provides a farnesyl diphosphate-producing
mutant prenyl diphosphate synthase which retains the properties
that were owned by the native prenyl diphosphate synthase.
The present invention also provides a DNA or an RNA encoding the
above enzyme.
The present invention further provides a recombinant vector and
more specifically an expression vector comprising the above
DNA.
The present invention further provides a host transformed by the
above vector.
The present invention further provides a process for producing
prenyl diphosphates having not more than 15 carbons comprising the
step wherein the above enzyme is brought into contact with a
substrate selected from the group consisting of isopentenyl
diphosphate, dimethylallyl diphosphate, and geranyl
diphosphate.
The present invention further provides a process of production of a
mutant enzyme according to any of claims 1 to 8, said method
comprising the steps of culturing the above host and of harvesting
the expression product from the culture.
BRIEF EXPLANATION OF THE DRAWINGS
FIG. 1 is a graph showing the regions (I) to (V) and the aspartic
acid-rich domain I of various prenyl diphosphate synthases. In the
figure, the sequence represents the amino acid sequence of
geranylgeranyl diphosphate synthase, and ATGERPYRS is the one
derived from Arabidopsis thaliana, LA15778.p from Lupinas albus,
CAGERDIS from Capsicum annuum, ATGGPSRP from Arabidopsis thaliana,
GGPS-pep from Sulfolobus acidocaldarius, SPCRT.pep from Rhodobactor
sphaeroides, RCPHSYNG from Rhodobactor capsulatus, EHCRTS.pe from
Erwinia herbicola, MXCRTNODA from Myxococcus thaliana, and
NCAL3.pep from Neurospora crassa. The number indicated on the left
of each amino acid sequence represents the site from the N-terminal
side of each geranylgeranyl diphosphate synthase at the N-terminal
of the amino acid sequence.
FIG. 2 is a graph showing the thermal stability of the mutant
prenyl diphosphate synthase. The ordinate shows the relative
activity to 100% at incubation at 60.degree. C. The abscissa shows
the incubation temperature. SacGGPS is the geranylgeranyl
diphosphate synthase prior to mutation. The others represent the
mutant type enzyme of each. BstFPS is the farnesyl diphosphate
synthase derived from Bacillus stearothermophillus.
FIG. 3 shows a photograph of a development pattern of thin layer
chromatography of the dephosphorylated reaction products of the
mutant prenyl diphosphate synthase when geranyl diphosphate was
used as the allylic substrate. In the figure, ori. represents the
origin of development, and s.f. represents the solvent front.
GOH is geraniol, FOH is farnesol, GGOH is geranyl geraniol, and
GFOH is geranylfarnesol, and these are produced from
dephosphorylation of geranyl diphosphate, farnesyl phosphate,
geranylgeranyl diphosphate, and geranylfarnesyl diphosphate,
respectively. SacGGPS is the geranylgeranyl diphosphate synthase
prior to mutation. The others are each mutant enzymes.
DETAILED DESCRIPTION
It has been proposed that there are five conserved regions in the
amino acid sequence of a prenyl diphosphate synthase (one subunit
in the case of a heterodimer) [A. Chem et al., Protein Science Vol.
3, pp. 600 607, 1994]. It is also known that of the five conserved
regions, there is an aspartic acid-rich domain conserved sequence I
[DDXX(XX)D] (wherein X denotes any amino acid, and the two X's in
the parentheses may not be present) in region II. Although there is
also an aspartic acid-rich domain indicated as "DDXXD" in region V,
the aspartic acid-rich domain used to specify the modified region
of the amino acid sequence of the present invention is the one
present in region II, and this domain is termed as the aspartic
acid-rich domain I as compared to the aspartic acid-rich domain II
present in region V.
As the prenyl diphosphate synthases having the aspartic acid-rich
domain as described above, there can be mentioned farnesyl
diphosphate synthase, geranylgeranyl diphosphate synthase,
hexaprenyl diphosphate synthase, heptaprenyl diphosphate synthase,
octaprenyl diphosphate synthase, nonaprenyl diphosphate synthase,
undecaprenyl diphosphate synthase, and the like. More specific
examples include the farnesyl diphosphate synthase of Bacillus
stearothermophilus, the farnesyl diphosphate synthase of
Escherichia coli, the farnesyl diphosphate synthase of
Saccharomyces cerevisiae, the farnesyl diphosphate synthase of the
rat, the farnesyl diphosphate synthase of the human, the
geranylgeranyl diphosphate synthase of Neurospora crassa, the
hexaprenyl diphosphate synthase of Saccharomyces cerevisiae, and
the like.
By way of example of some of these, regions I to V and the aspartic
acid-rich domain I (in the box) in region II of the amino acid
sequence of geranylgeranyl diphosphate synthases are shown in FIG.
1.
The present invention can be applied to any prenyl diphosphate
synthase having the aspartic acid-rich domain I.
In accordance with the present invention, in the amino acid
sequence of a prenyl diphosphate synthase, at least one amino acid
residue selected from (a) the amino acid residues in between the
amino acid residue located at the fifth position in the N-terminal
direction from D of the N-terminal and the amino acid residue
located at the first position in the N-terminal direction from D of
said N-terminal of the aspartic acid-rich domain DDXX(XX)D (wherein
X denotes any amino acid, and the two X's in the parentheses may
not be present) present in region II, and (b) the amino acid
residue located at the first position in the N-terminal direction
from D of the C-terminal of said aspartic acid-rich domain has been
substituted by another amino acid, and/or
an additional one or more amino acids have been inserted in between
the amino acid residue located at the first position in the
N-terminal side from D of the C-terminal and D of said C-terminal
of said aspartic acid-rich domain.
The mutant prenyl diphosphate synthase of the present invention can
synthesize a farnesyl diphosphate having a shorter chain length
than the prenyl diphosphate synthesized by the native prenyl
diphosphate synthase.
In accordance with the present invention, by way of example, the
gene of the geranylgeranyl diphosphate synthase of a highly
thermophilic archaea, Sulfolobus acidocaldarius, is used as the
starting material. Sulfolobus acidocaldarius is available from ATCC
as ATCC No. 33909. The method for cloning the gene has been
described in detail in Japanese Unexamined Patent Publication No.
7-308193. It has also been disclosed with the accession No. D28748
in the gene information data base such as GenBank. By using the
sequence it can be cloned in the conventional method known in the
art. An example of the other cloning methods is illustrated in
Example 1 herein and its nucleotide sequence is shown as SEQ ID No:
2.
More specifically, the mutant enzyme of the present invention is a
mutant prenyl diphosphate synthase characterized in that at least
one amino acid selected from phenylalanine in position 77,
threonine at position 78, valine at position 80, histidine at
position 81, and isoleucine at position 84 has been substituted by
another amino acid, and/or amino acid(s) have been inserted in
between isoleucine at position 84 and methionine at position 85 in
the geranylgeranyl diphosphate synthase having the amino acid
sequence as set forth in SEQ ID No: 1.
By way of example, there is provided the amino acid sequences
wherein the amino acids have been substituted as shown below:
Mutant enzyme 1: Changes from threonine at position 78 to
phenylalanine, and histidine at position 81 to alanine;
Mutant enzyme 2: Changes from threonine at position 78 to
phenylalanine, and histidine at position 81 to leucine;
Mutant enzyme 3: Changes from phenylalanine at position 77 to
tyrosine, threonine at position 78 to phenylalanine, and histidine
at position 81 to leucine;
Mutant enzyme 4: Changes from phenylalanine at position 77 to
tyrosine, threonine at position 78 to phenylalanine, and histidine
at position 81 to alanine;
Mutant enzyme 5: Changes from phenylalanine at position 77 to
tyrosine, threonine at position 78 to serine, valine at position 80
to isoleucine, and isoleucine at position 84 to leucine, and an
insertion of proline and serine in between isoleucine at position
84 and methionine at position 85.
In accordance with the present invention, it is indicated that the
mutant prenyl diphosphate synthase retains the characteristic
properties that were owned by the native prenyl diphosphate
synthase. By way of example, the above-mentioned five mutant
enzymes show thermo resistance almost equal to that owned by the
native geranylgeranyl diphosphate synthase.
It is known that an enzyme can sometimes exhibit its original
enzymatic activity even when it has been modified by addition,
removal, and/or substitution of one or a few amino acids as
compared to the original amino acid sequence. Therefore, the
present invention is intended to encompass those enzymes that have
been modified by addition, deletion, and/or substitution of one or
a few, for example up to five, or up to 10, amino acids as compared
to the amino acid sequence as set forth in SEQ ID No: 1 and that
can perform its original function.
The present invention also provides the genes encoding various
mutant enzymes mentioned above, the vectors containing those genes,
specifically expression vectors, and the hosts transformed by said
vectors. The gene (DNA) of the present invention can be readily
obtained, for example, by introducing mutation into the DNA
encoding the original amino acid sequence as set forth in SEQ ID
No: 1 using a conventional method such as site-directed
mutagenesis, PCR and the like.
Furthermore, once the amino acid sequence of the desired enzyme has
been determined, an appropriate nucleotide sequence encoding it can
be determined, and the DNA can be chemically synthesized in
accordance with a conventional method of DNA synthesis.
The present invention further provides an expression vector
comprising DNA such as the one mentioned above, the host
transformed by said expression vector, and a method for producing
the enzyme or peptide of the present invention using these
hosts.
Expression vectors contain an origin of replication, expression
regulatory sequences etc., but they may differ depending on hosts
used. As the hosts, there are mentioned procaryotes, for example,
bacteria such as Escherichia coli, organisms of genus Bacillus such
as Bacillus subtilis, and eukaryotic microorganisms, for example,
fungi, for example yeast, for example Saccharomyces cerevisiae of
genus Saccharomyces and Pichia pastoris of genus Pichia,
filamentous fungi, for example the genus Asperaillus such as
Asperaillus niger, animal cells, for example the cultured cells of
the silkworm, cultured cells of higher animals, for example CHO
cells, and the like. Furthermore, plants may also be used as the
host.
As set forth in Examples, in accordance with the present invention,
by cultivating the host transformed by the DNA of the present
invention, farnesyl diphosphates may be accumulated in the culture
broth, which may be harvested to produce their farnesyl
diphosphates. Furthermore, in accordance with the invention,
farnesyl diphosphates may also be produced by contacting the mutant
prenyl diphosphate synthase produced by the method of the invention
to the substrate isopentenyl diphosphate and each allyl substrate
such as dimethylallyl diphosphate and geranyl diphosphate.
When Escherichia coli is used as the host, it is known that the
host has the regulatory functions at the stage of transcribing mRNA
from DNA and of translating protein from mRNA. As the promoter
sequence regulating mRNA synthesis, in addition to the naturally
occurring sequences (for example, lac, trp, bla, lpp, P.sub.L,
P.sub.R, ter, T3, T7, etc.), there are known their mutants (for
example, lac UV5), and the sequences (such as tac, trc, etc.) in
which a naturally occurring promoter is artificially fused, and
they can be used for the present invention.
It is known that the distance between the sequence of the ribosome
.[.biding.]. .Iadd.binding .Iaddend.site (GGAGG and similar
sequences thereof) and the initiation codon ATG is important as the
sequence regulating the ability of synthesizing protein from mRNA.
It is also well known that a terminator (for example, a vector
containing rrn PT.sub.1 T.sub.2 is commercially available from
Pharmacia) that directs transcription termination at the 3'-end
affects the efficiency of protein synthesis by a recombinant.
As the vectors that can be used for preparation of the recombinant
vectors of the present invention, commercially available vectors
are used as they are, or various vectors may be mentioned that are
derived depending on the intended use. For example, there can be
mentioned pBR322, pBR327, pKK223-3, pKK233-3, pTrc99, and the like
having a replicon derived from pMB1; pUC18, pUC19, pUC118, pUC119,
pTV118N, pTV119N, pBluescript, pHSG298, pHSG396, and the like that
have been altered to enhance copy numbers; and pACYC177, pACYC184,
and the like that have a replicon derived from p15A; and,
furthermore, plasmids derived from pSC101, ColE1, R1, F factor, and
the like.
Furthermore, fusion protein-expressing vectors that enable easier
purification such as pGEX-2T, pGEX-3X, pMal-c2 may be used. One
example of the gene used as the starting material of the present
invention has been described in Japanese Unexamined Patent
Publication No. 7-308193.
Furthermore, in addition to plasmids, virus vectors such as .lamda.
phage or M13 phage, or transposon may be used for the
transformation of genes. With regard to the transformation of the
gene into microorganisms other than Escherichia coli, gene
transformation into organisms of genus Bacillus by pUB110
(commercially available from Sigma) or pHY300PLK (commercially
available from Takara Shuzo) is known. These vectors are described
in "Molecular Cloning" (J. Sambrook, E. F. Fritsch, and T.
Maniatis, Cold Spring Harbor Laboratory Press) and "Cloning Vector"
(P. H. Pouwels, B. B. Enger, Valk, and W. J. Brammar, Elsevier),
and catalogues of the manufacturers.
Integration of the DNA fragment encoding the prenyl diphosphate
synthase and, where needed, the DNA fragment having the function of
regulating expression of the gene of said enzyme into these vectors
can be performed by a known method using an appropriate restriction
enzyme and ligase. Specific examples of the plasmids thus
constructed include, for example, pBs-SacGGPS.
As the microorganisms into which genes can be directly introduced
using such recombinant vectors include Escherichia coli and
microorganisms of the genus Bacillus. Such transformation can also
be carried out using general method, for example the CaCl.sub.2
method and the protoplast method as described in "Molecular
Cloning" (J. Sambrook, E. F. Fritsch, and T. Maniatis, Cold Spring
Harbor Laboratory Press) and "DNA Cloning" Vol. I to III (D. M.
Clover ed., IRL PRESS).
In order to produce the mutant enzyme of the present invention, a
host transformed as above is cultured, and then said culture is
subjected to any method comprising salting out, precipitation with
an organic solvent, gel chromatography, affinity chromatography,
hydrophobic chromatography, ion exchange chromatography, and the
like to recover and purify said enzyme.
The present invention also provides a process for producing
farnesyl diphosphates using the enzyme of the present invention.
According to this process, the enzyme of the present invention is
reacted with a substrate in a medium, particularly an aqueous
medium, and then, as desired, the prenyl diphosphate is harvested
from the reaction medium. As the enzyme, not only a purified enzyme
but also a crude enzyme that may be semi-purified to various
stages, or a mixture of the cultured broth of a microorganism may
be used. Alternatively there may be used immobilized enzymes
prepared according to the general method from said enzyme, said
crude enzyme, or a product containing the enzyme.
As the substrate, there may be used dimethyl allyl diphosphates or
geranyl diphosphates and isopentenyl diphosphates. As the reaction
medium, water or an aqueous buffer solution, for example Tris
buffer or phosphate buffer and the like, may be used.
By using the method of producing the mutant prenyl diphosphate
synthase obtained by the present invention, the mutant prenyl
diphosphate synthase derived from .[.a.]. .Iadd.an .Iaddend.archaea
may be created that is more stable and thus easier to handle and
that produces .[.prrenyl.]. .Iadd.prenyl .Iaddend.diphosphate.
Furthermore, there is also expected a creation of the farnesyl
diphosphate-producing mutant prenyl diphosphate synthase that has
the property of the prenyl diphosphate synthase prior to mutation
(for example, salt stability or stability in a wide range of pH)
added thereto.
In the claims and the specification of the present invention, amino
acid residues are expressed by the one-letter codes or three-letter
codes as described hereinbelow:
A; Ala; alanine
C; Cys; cysteine
D; Asp; aspartic acid
E; Glu; glutamic acid
F; Phe; phenylalanine
G; Gly; glycine
H; His; histidine
I; Ile; isoleucine
K; Lys; lysine
L; Leu; leucine
M; Met; methionine
N; Asn; asparagine
P; Pro; proline
Q; Gln; glutamine
R; Arg; arginine
S; Ser; serine
T; Thr; threonine
V; Val; valine
W; Trp; tryptophan
Y; Tyr; tyrosine
Substitution of amino acid is expressed in the order of "the amino
acid residue before substitution," "number of the amino acid
residue," and "the amino acid residue after substitution," by the
one-letter codes of amino acids. For example, the mutation in which
a tyrosine residue at position 81 is replaced with a methionine
residue is expressed as Y81M. Furthermore, the insertion of amino
acid residues is expressed by "the number of the amino acid residue
at the N-terminal side of the insertion site prior to insertion,"
"the amino acid residue that was inserted," and "the number of the
amino acid residue at the C-terminal side of the insertion site
prior to insertion." For example, the insertion of alanine in
between the amino acid at position 84 and the amino acid at
position 85 is expressed as 84A85.
EXAMPLES
The present invention is now explained with reference to specific
examples, but they must not be construed to limit the invention in
any way.
Example 1
Construction of a Plasmid Containing the Gene for
.[.Geranylaeranyl.]. .Iadd.Geranylgeranyl .Iaddend.Diphosphate
Synthase
The gene for the geranylgeranyl diphosphate synthase (hereinafter
referred to as SacGGPS) derived from Sulfolobus acidocaldarius was
subcloned at the HindIII site of the plasmid vector pBluescript II
(KS+) commercially available from Toyoboseki. The plasmid DNA was
designated as pBs-SacGGPS. The SacGGPS gene is available from
Escherichia coli DH5.alpha. (pGGPS1) that was internationally
deposited on Jan. 31, 1994 with the National Institute of
Bioscience and Human Technology Agency of Industrial Science and
Technology, of Ibalaki, Japan under the accession number of FERM
BP-4982.
Also, the entire nucleotide sequence of the SacGGPS gene has been
published in Japanese Unexamined Patent Publication No. 7-308193
Shin-ichi Ohnuma et al. (1994) The Journal of Biological Chemistry
Vol. 269:14792 14797, or in the genetic information data bank such
as GenBank under the accession number D28748. Since Sulfolobus
acidocaldarius is also available from various depositories of
microorganisms such as ATCC etc. (as ATCC No. 33909), the DNA of
the gene region of SacGGPS can be obtained by the conventional gene
cloning method.
Example 2
Synthesis of the Oligonucleotides for Introducing Mutation
For introducing mutation of the gene of geranylgeranyl diphosphate
synthase, the following oligonucleotides were designed and
synthesized:
Primer DNA (T78F, H81A): 5'-CATACTTTTTTCCTTGTGGCTGATGATATCATG
GATC-3' (SEQ ID No: 3)
Primer DNA (T78F, H81L): 5'-CATACTTTTTTCCTTGTGCTTGATGATATCATG
GATC-3' (SEQ ID No: 4)
Primer DNA (F77Y, T78F, H81L): 5'-CATACTTATTTCCTTGTGCTTGATGATATCAT
GGATC-3' (SEQ ID No: 5)
Primer DNA (F77Y, T78F, H81A): 5'-CATACTTATTTCCTTGTGGCTGATGATATCAT
GGATC-3' (SEQ ID No: 6)
Primer DNA (F77Y, T78S, V80I, I84L, 84PS85):
5'-GTTCTTCATACTTATTCGCTTATTCATGATAGT ATT-3' (SEQ ID No: 7), and
5'-ATTCATGATGATC TTCCATCGATGGATCAAGAT-3' (SEQ ID No: 8).
Introduction of the mutation (F77Y, T78S, V80I, I84L, 84PS85) was
effected using two nucleotides. First, mutation was introduced as
mentioned in Example 3 using the oligonucleotide
5'-GTTCTTCATACTTATTCGCTTATTCATGATAG.[.TATT-31.]. .Iadd.TATT-3'
.Iaddend.(SEQ ID No: 7) and a transformant was prepared in
accordance with Example 4, and furthermore mutation was introduced
into the plasmid thus obtained using the oligonucleotide
5'-ATTCATGATGATCTTCCATCGATGGATCAAGAT-3' (SEQ ID No: 8).
These nucleotides have a mutation in the codon encoding at least
one amino acid residue selected from phenylalanine at position 77,
threonine at position 78, valine at position 80, histidine at
position 81, and isolcucine at position 84 in SacGGPS. In addition
to the introduction of the codon encoding an amino acid that has
been inserted in between isoleucine at position 84 and methionine
at position 85, they are designed to newly introduce the cleavage
site of the restriction enzyme BspHI (5'TGATGA3'), the cleavage
site of the restriction enzyme EcoRV (5'GATATC3'), or the cleavage
site of the restriction enzyme ClaI (5'ATCGAT3'). In the
introduction of the cleavage site of BspHI, the amino acid sequence
encoded by the SacGGPS gene does not change due to degeneracy of
codons, or it is a site for an introduction of mutation. This is
used to detect the substitution-mutated plasmid by means of agarose
gel electrophoresis after digestion with an appropriate restriction
enzyme, since the introduction of mutation by substitution into the
SacGGPS gene simultaneously produces new cleavage sites of
restriction enzymes.
These primer DNA's were subjected to phosphorylation treatment at
37.degree. C. for 30 minutes in the reaction medium shown below
followed by denaturation at 70.degree. C. for 10 minutes: 10
pmol/.mu.l primer DNA 2 .mu.l 10.times.kination buffer 1 .mu.l 10
mM ATP 1 .mu.l .[.H2O.]. .Iadd.H.sub.2O .Iaddend.5 .mu.l T4
polynucleotide kinase 1 .mu.l wherein the 10.times.kination buffer
is 1000 mM Tris-Cl (pH 8.0), 100 mM MgCl.sub.2, and 70 mM DTT.
Example 3
The Introduction of Substitution-Mutation of the SacGGPSS Gene
Using each primer DNA constructed in Example 2,
substitution-mutation was introduced into the plasmid prepared in
Example 1 in accordance with the Kunkel method. Mutan-K kit
commercially available from Takara Shuzo was used to perform the
Kunkel method. The experimental procedure was as described in the
kit insert. The substitution-mutation of the plasmid need not be
conducted by the Kunkel method. For example, an identical result
can be obtained by a method using the polymerase chain reaction
(PCR).
Using Escherichia coli CJ236 in the Mutan-K kit as the host cell, a
single strand DNA was obtained in which a thymine base in plasmid
pBS-SacGGPS was replaced with a deoxyuracil base.
The single strand DNA thus obtained was used as the template in the
reaction in which a primer DNA for synthesizing a complementary
strand was treated in the following reaction solution at 65.degree.
C. for 15 minutes and then annealed by allowing to stand at
37.degree. C. for 15 minutes: Single strand DNA 0.6 pmol Annealing
buffer solution 1 .mu.l Primer DNA solution (Example 2) 1 .mu.l
.[.H2O.]. .Iadd.H.sub.2O .Iaddend.make to a final volume of 10
.mu.l in which the annealing buffer solution is 200 mM Tris-Cl (pH
8.0), 100 mM MgCl.sub.2, 500 MM NaCl, and 10 mM DTT.
Furthermore, 25 .mu.l of the elongation buffer solution, 60 units
of Escherichia coli DNA ligase, and 1 unit of T4 DNA polymerase
were added to synthesize the complementary strands at 25.degree. C.
for 2 hours. The elongation buffer solution is 50 mM Tris-Cl (pH
8.0), 60 mM ammonium acetate, 5 mM MgCl.sub.2, 5 mM DTT, 1 mM NAD,
and 0.5 mM dNTP.
After the reaction is over, 3 .mu.l of 0.2 M EDTA (pH 8.0) was
added thereto and was subjected to treatment at 65.degree. C. for 5
minutes to stop the reaction.
Example 4
Construction of a Recombinant Having a Gene in Which
Substitution-Mutation Has Been Introduced into the SacGGPS Gene
The DNA solution constructed in accordance with Example 3 was used
to transform Escherichia coli XL1-Blue by the CaCl.sub.2 method. An
alternative method such as electroporation gives a similar result.
A host cell other than Escherichia coli XL1-Blue, for example JM109
and the like also gave a similar result.
The transformant obtained by the CaCl.sub.2 method was plated onto
the agar plate containing ampicillin, a selectable marker of
transformants, and was incubated overnight at 37.degree. C.
Of the transformants obtained as above, the substitution-mutated
pBs-SacGGPS plasmid that has a cleavage site of BspHI, EcoRV or
ClaI was selected. The nucleotide sequence in the neighborhood of
the codon corresponding to the amino acid residue that undergoes
mutation of the SacGGPS gene of the selected substitution-mutated
pBs-SacGGPS plasmid was determined by the dideoxy method. As a
result, the pBs-SacGGPS plasmid containing the following five
mutated SacGGPS genes was obtained. The nucleotide sequences
encoding the amino acid sequences from the amino acid at position
77 to the amino acid at position 85 is shown below:
Mutation Nucleotide sequence T77F, H81A:
5'-TTTTTCCTTGTGGCTGATGATATCATG-3' (SEQ ID No: 9) T78P, H81L:
5'-TTTTTCCTTGTGCTTGATGATATCATG-3' (SEQ ID No: 10) F77Y, T78F, H81L:
5'-TATTTCCTTGTGCTTGATGATATCATG-.[.31.]. .Iadd.3'.Iaddend. (SEQ ID
No: 11) F77Y, T78F, H81A: 5'-TATTTCCTTGTGGCTGATGATATCATG-3' (SEQ ID
No: 12) F77Y, T78S, V80I, I84L, 84PS85:
5'-TATTCGCTTATTCATGATGATCTTCCATCGATG-3' (SEQ ID No: 13) Wild type:
5'-TTTACGCTTGTGCATGATGATATTATG-3' (SEQ ID No: 14).
Example 5
Measurement of Activity of the Mutant Prenyl Diphosphate
Synthase
Crude enzyme solutions were prepared as follows from 6
transformants comprising 5 mutant SacGGPS genes and one wild type
SacGGPS gene obtained in Example 4.
The transformant cultured overnight in the 2.times.LB medium was
centrifuged to harvest cells, and then the cells were suspended
into a buffer for cell homogenization (50 mM calcium phosphate
buffer solution (pH 5.8), 10 mM .beta.-mercaptoethanol, 1 mM EDTA).
This was homogenized by sonnication and then centrifuged at
4.degree. C. at 10,000 r.p.m. for 10 minutes. The supernatant
obtained was treated at 55.degree. C. for 12 hours to inactivate
the activity of prenyl diphosphate synthase derived from
Escherichia coli. This was further centrifuged under the same
condition and the supernatant obtained was used as a crude enzyme
extract. When thermo stability was investigated the enzyme extract
was incubated at 60.degree. C., 70.degree. C., or 80.degree. C.
(60.degree. C., 65.degree. C., 67.degree. C., or 70.degree. C. for
the enzymes derived from Bacillus stearothermophilus) for one hour
prior to reaction. The reaction was conducted at 55.degree. C. for
15 minutes in the following reaction solution:
[1-.sup.14C]-isopentenyl diphosphate (1 Ci/mol) 25 nmol Allylic
diphosphate (geranyl diphosphate) 25 nmol Potassium phosphate
buffer (pH 5.8) 10 mM MgCl.sub.2 5 mM Enzyme solution 100 .mu.g
H.sub.2O to make 200 .mu.l
After the reaction is over, 200 .mu.l of saturated NaCl was added
to the reaction solution and 1 ml of water-saturated butanol was
added thereto, which was then agitated, centrifuged, and separated
into two phases. To 800 .mu.l of the butanol layer obtained was
added 3 ml of a liquid scintillator and then the radioactivity was
measured by the scintillation counter. The result is shown in FIG.
2.
The mutant prenyl diphosphate synthase has exhibited a thermo
stability which is equal to that of the native geranylgeranyl
diphosphate synthase, and is higher than that of the farnesyl
diphosphate synthase derived from Bacillus stearothermophilus.
The solvent is evaporated from the remainder of the butanol layer
by purging nitrogen gas thereinto while heating the layer in order
to concentrate to a volume of about 0.5 ml. To the concentrate were
added 2 ml of methanol and one ml of potato acid phosphatase
solution (2 mg/ml potato acid phosphatase, 0.5 M sodium acetate (pH
4.7)) to effect the dephosphorylation reaction at 37.degree. C.
Subsequently the dephosphorylated reaction product was extracted
with 3 ml of n-pentane.
This was concentrated by evaporating the solvent by purging
nitrogen gas thereinto, which was then analyzed by TLC (reverse
phase TLC plate: LKC18 (Whatman), development solvent:
acetone/water=9/1). The developed dephosphorylated reaction product
was analyzed by the Bio Image Analyzer BAS2000 (Fuji Photo Film) to
determine the location of radioactivity. The result when geranyl
diphosphate was used as the allylic substrate is shown in FIG.
3.
The reaction product of the mutant prenyl diphosphate synthase was
shown to be a farnesyl diphosphate.
SEQUENCE LISTINGS
1
141330PRTSulfolobus acidocaldarius 1Met Ser Tyr Phe Asp Asn Tyr Phe
Asn Glu Ile Val Asn Ser Val Asn 1 5 10 15Asp Ile Ile Lys Ser Tyr
Ile Ser Gly Asp Val Pro Lys Leu Tyr Glu 20 25 30Ala Ser Tyr His Leu
Phe Thr Ser Gly Gly Lys Arg Leu Arg Pro Leu 35 40 45Ile Leu Thr Ile
Ser Ser Asp Leu Phe Gly Gly Gln Arg Glu Arg Ala 50 55 60Tyr Tyr Ala
Gly Ala Ala Ile Glu Val Leu His Thr Phe Thr Leu Val65 70 75 80His
Asp Asp Ile Met Asp Gln Asp Asn Ile Arg Arg Gly Leu Pro Thr 85 90
95Val His Val Lys Tyr Gly Leu Pro Leu Ala Ile Leu Ala Gly Asp Leu
100 105 110Leu His Ala Lys Ala Phe Gln Leu Leu Thr Gln Ala Leu Arg
Gly Leu 115 120 125Pro Ser Glu Thr Ile Ile Lys Ala Phe Asp Ile Phe
Thr Arg Ser Ile 130 135 140Ile Ile Ile Ser Glu Gly Gln Ala Val Asp
Met Glu Phe Glu Asp Arg145 150 155 160Ile Asp Ile Lys Glu Gln Glu
Tyr Leu Asp Met Ile Ser Arg Lys Thr 165 170 175Ala Ala Leu Phe Ser
Ala Ser Ser Ser Ile Gly Ala Leu Ile Ala Gly 180 185 190Ala Asn Asp
Asn Asp Val Arg Leu Met Ser Asp Phe Gly Thr Asn Leu 195 200 205Gly
Ile Ala Phe Gln Ile Val Asp Asp Ile Leu Gly Leu Thr Ala Asp 210 215
220Glu Lys Glu Leu Gly Lys Pro Val Phe Ser Asp Ile Arg Glu Gly
Lys225 230 235 240Lys Thr Ile Leu Val Ile Lys Thr Leu Glu Leu Cys
Lys Glu Asp Glu 245 250 255Lys Lys Ile Val Leu Lys Ala Leu Gly Asn
Lys Ser Ala Ser Lys Glu 260 265 270Glu Leu Met Ser Ser Ala Asp Ile
Ile Lys Lys Tyr Ser Leu Asp Tyr 275 280 285Ala Tyr Asn Leu Ala Glu
Lys Tyr Tyr Lys Asn Ala Ile Asp Ser Leu 290 295 300Asn Gln Val Ser
Ser Lys Ser Asp Ile Pro Gly Lys Ala Leu Lys Tyr305 310 315 320Leu
Ala Glu Phe Thr Ile Arg Arg Arg Lys 325 3302993DNASulfolobus
acidocaldariusCDS(1)...(993) 2atg agt tac ttt gac aac tat ttt aat
gag att gtt aat tct gta aac 48Met Ser Tyr Phe Asp Asn Tyr Phe Asn
Glu Ile Val Asn Ser Val Asn 1 5 10 15gac att att aag agc tat ata
tct gga gat gtt cct aaa cta tat gaa 96Asp Ile Ile Lys Ser Tyr Ile
Ser Gly Asp Val Pro Lys Leu Tyr Glu 20 25 30gcc tca tat cat ttg ttt
aca tct gga ggt aag agg tta aga cca tta 144Ala Ser Tyr His Leu Phe
Thr Ser Gly Gly Lys Arg Leu Arg Pro Leu 35 40 45atc tta act ata tca
tca gat tta ttc gga gga cag aga gaa aga gct 192Ile Leu Thr Ile Ser
Ser Asp Leu Phe Gly Gly Gln Arg Glu Arg Ala 50 55 60tat tat gca ggt
gca gct att gaa gtt ctt cat act ttt acg ctt gtg 240Tyr Tyr Ala Gly
Ala Ala Ile Glu Val Leu His Thr Phe Thr Leu Val 65 70 75 80cat gat
gat att atg gat caa gat aat atc aga aga ggg tta ccc aca 288His Asp
Asp Ile Met Asp Gln Asp Asn Ile Arg Arg Gly Leu Pro Thr 85 90 95gtc
cac gtg aaa tac ggc tta ccc tta gca ata tta gct ggg gat tta 336Val
His Val Lys Tyr Gly Leu Pro Leu Ala Ile Leu Ala Gly Asp Leu 100 105
110cta cat gca aag gct ttt cag ctc tta acc cag gct ctt aga ggt ttg
384Leu His Ala Lys Ala Phe Gln Leu Leu Thr Gln Ala Leu Arg Gly Leu
115 120 125cca agt gaa acc ata att aag gct ttc gat att ttc act cgt
tca ata 432Pro Ser Glu Thr Ile Ile Lys Ala Phe Asp Ile Phe Thr Arg
Ser Ile 130 135 140ata att ata tcc gaa gga cag gca gta gat atg gaa
ttt gag gac aga 480Ile Ile Ile Ser Glu Gly Gln Ala Val Asp Met Glu
Phe Glu Asp Arg145 150 155 160att gat ata aag gag cag gaa tac ctt
gac atg atc tca cgt aag aca 528Ile Asp Ile Lys Glu Gln Glu Tyr Leu
Asp Met Ile Ser Arg Lys Thr 165 170 175gct gca tta ttc tcg gca tcc
tca agt ata ggc gca ctt att gct ggt 576Ala Ala Leu Phe Ser Ala Ser
Ser Ser Ile Gly Ala Leu Ile Ala Gly 180 185 190gct aat gat aat gat
gta aga ctg atg tct gat ttc ggt acg aat cta 624Ala Asn Asp Asn Asp
Val Arg Leu Met Ser Asp Phe Gly Thr Asn Leu 195 200 205ggt att gca
ttt cag att gtt gac gat atc tta ggt cta aca gca gac 672Gly Ile Ala
Phe Gln Ile Val Asp Asp Ile Leu Gly Leu Thr Ala Asp 210 215 220gaa
aag gaa ctt gga aag cct gtt ttt agt gat att agg gag ggt aaa 720Glu
Lys Glu Leu Gly Lys Pro Val Phe Ser Asp Ile Arg Glu Gly Lys225 230
235 240aag act ata ctt gta ata aaa aca ctg gag ctt tgt aaa gag gac
gag 768Lys Thr Ile Leu Val Ile Lys Thr Leu Glu Leu Cys Lys Glu Asp
Glu 245 250 255aag aag att gtc cta aag gcg tta ggt aat aag tca gcc
tca aaa gaa 816Lys Lys Ile Val Leu Lys Ala Leu Gly Asn Lys Ser Ala
Ser Lys Glu 260 265 270gaa tta atg agc tca gca gat ata att aag aaa
tac tct tta gat tat 864Glu Leu Met Ser Ser Ala Asp Ile Ile Lys Lys
Tyr Ser Leu Asp Tyr 275 280 285gca tac aat tta gca gag aaa tat tat
aaa aat gct ata gac tct tta 912Ala Tyr Asn Leu Ala Glu Lys Tyr Tyr
Lys Asn Ala Ile Asp Ser Leu 290 295 300aat caa gtc tcc tct aag agt
gat ata cct gga aag gct tta aaa tat 960Asn Gln Val Ser Ser Lys Ser
Asp Ile Pro Gly Lys Ala Leu Lys Tyr305 310 315 320cta gct gaa ttt
acg ata aga agg aga aaa taa 993Leu Ala Glu Phe Thr Ile Arg Arg Arg
Lys * 325 330337DNASulfolobus acidocaldarius 3catacttttt tccttgtggc
tgatgatatc atggatc 37437DNASulfolobus acidocaldarius 4catacttttt
tccttgtgct tgatgatatc atggatc 37537DNASulfolobus acidocaldarius
5catacttatt tccttgtgct tgatgatatc atggatc 37637DNASulfolobus
acidocaldarius 6catacttatt tccttgtggc tgatgatatc atggatc
37736DNASulfolobus acidocaldarius 7gttcttcata cttattcgct tattcatgat
agtatt 36833DNASulfolobus acidocaldarius 8attcatgatg atcttccatc
gatggatcaa gat 33927DNASulfolobus acidocaldarius 9tttttccttg
tggctgatga tatcatg 271027DNASulfolobus acidocaldarius 10tttttccttg
tgcttgatga tatcatg 271127DNASulfolobus acidocaldarius 11tatttccttg
tgcttgatga tatcatg 271227DNASulfolobus acidocaldarius 12tatttccttg
tggctgatga tatcatg 271333DNASulfolobus acidocaldarius 13tattcgctta
ttcatgatga tcttccatcg atg 331427DNASulfolobus acidocaldarius
14tttacgcttg tgcatgatga tattatg 27
* * * * *
References