The nucleotide sequence of a DNA coding for ectoine synthase has been determined by isolating a gene coding for an enzyme involved in the biosynthesis of ectoine in the form of a DNA comprising about 4.2 kilobase pairs which is obtained from Halomonas sp. KS-3 by cleaving with restriction endonucleases EcoRI and SalI. By introducing the obtained gene DNA into E. coli, it is possible to provide the capability of biosynthetizing ectoine and the characteristic of high osmotic tolerance, thus permitting the development of an efficient fermentation technique utilizing the resultant transformant and the creation of plants having high resistance to drought.

TECHNICAL FIELD 
This invention relates to a DNA coding for an enzyme involved in the 
biosynthesis of ectoine, a method for giving ectoine synthetic ability to 
a host cell by introducing said DNA thereto, and a transformed host cell. 
PRIOR ART 
Under an environment of a high osmotic pressure, a certain microorganisms 
can obtain a tolerance to the surrounding stress by accumulating so-called 
"compatible solute" within their cells. It is known that the compatible 
solute includes saccharides, polyols, betaines or a certain amino acids 
(cf. Truper, H. G. et al., Experientia, Vol. 42, pp. 1182-1187, 1986). 
Ectoine is a cyclic amino acid, which is 
1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid or 
3,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid. Said ectoine has 
been found as a compatible solute which is produced by a halophilic 
microorganism, Ectothiorhodospira halochloris, and it is further known 
that it has a tolerance to a high osmotic pressure ("high osmotic 
tolerance") cf. Galinski, E. A. et al., Eur. J. Biochem., Vol. 149, pp. 
135-139, 1985; Mitsuo Takano et al., a program for Symposium of The Japan 
Fermentation Engineering Association, p. 193, 1988!. There are also some 
reports as to the biosynthetic pathway of ectoine (cf. Peters, P. et al., 
FEMS Microbiol. Lett., Vol. 71, pp. 157-162, 1990) and as to the physical 
properties thereof (cf. Khunajakr, N., et al., Annual Reports of 
International Center of Cooperative Research in Biotechnology, Japan, Vol. 
12, pp. 157-167, 1989). 
Ectoine is biosynthetized from L-aspartate-.beta.-semialdehyde, in which 
steps L-diaminobutyric acid trans-aminase, L-diaminobutyric 
acetyltransferase, and ectoine synthase are involved. Hereinafter, these 
enzymes are generally called as "ectoine synthetases". As a system being 
capable of inducing the biosynthesis of an enzyme by bacteria, there is 
known so-called "operon" which is a regulatory system for expression of a 
gene, where a series of enzymes are synchronously induced by a single 
regulatory gene. The ectoine synthase is an enzyme which can synthetize 
ectoine from a-N-acetyl-diaminobutyric acid, and there are reports 
concerning a method for isolating and purifying said enzyme from a 
halophilic microorganism and the properties thereof (cf. Mihoko Yamamoto, 
et al., Summary of Symposium of Japan Biotechnology Society, p. 203, 
1992). However, the gene structure and nucleotide sequence of the ectoine 
synthase and the ectoine synthetases have never been known. 
On the basis of a knowledge that a certain micro-organism can accumulate 
ectoine within the cells thereof and thereby can grow even under an 
environment condition of strong stress to living bodies (e.g. high osmotic 
pressure), it has been proposed a method for extracting and isolating 
ectoine by aiming at the function of ectoine (cf. Khunajakr, N. et al., 
Annual Reports of International Center of Cooperative Research in 
Biotechnology, Japan, Vol. 12, pp. 157-167, 1989) and a method for 
chemical synthesis of ectoine (cf. JP-A-3-31265). 
DISCLOSURE OF THE INVENTION 
Aiming at the specific function of the ectoine, the present inventors have 
intensively investigated the ectoine from the following viewpoints. Where 
a high osmotic tolerance is given to, for example, a microorganism or a 
plant by giving an ability of biosynthesis of ectoine, not to ectoine per 
se, it will be able to develop a method for the efficient production of an 
useful product by a fermentation in a high concentration, and further it 
will be able to create a plant having a resistance to drought and having 
tolerance to a high osmotic pressure due to the droughty circumstance. 
Moreover, where the microorganisms are grown in a droughty ground such as 
in a desert, it will be also possible to change to a fertilized soil and 
further to create a plant which is suitable for planting in a droughty 
ground and at highly salty environment such as at a seaside.

BEST MODE FOR CARRYING OUT THE INVENTION 
Aiming at the existence of bacteria which can grow under a highly salty 
environment and can accumulate ectoine within the cells and thereby can 
exhibit high tolerance to a high osmotic pressure, the present inventors 
have looked for and have collected a bacteria having such characteristics 
from a soil at a northeastern area in Tailand and identified it. The 
bacteria having a high osmotic tolerance is classified as a Gram negative, 
aerobic bacillus of the genus Halomonas, which is positive in catalase, GC 
of DNA: 65.4-65.7 mol.%, and grows in a concentration of sodium chloride 
of from 0.3% to 24% (cf. Mitsuyoshi Okuda, et al., Summary of Symposium of 
Halophilic Microorganisms Research Association, Vol. 25, pp. 14-17, 1988). 
This bacteria was designated as "Halomonas sp. KS-3" and deposited to 
National Institute of Bioscience and Human-Technology, Agency of 
Industrial Science and Technology under Budapest Treaty with an accession 
number of FERM BP-4841 (accepted on Oct. 20, 1994) which has originally 
been deposited as an accession number of FERP P-13952 (accepted on Nov. 5, 
1993). 
It has been confirmed by the inventors that the Halomonas sp. KS-3 strain 
can produce ectoine (in accordance with the method as described in 
Khunajakr, N. et al., Annual Reports of International Center of 
Cooperative Research in Biotechnology, Japan, Vol. 12, pp. 157-167, 1989) 
and then ectoine synthase has been isolated from said culture cells. That 
is, the culture cells are lyzed with lysozyme, and after removing the 
nucleic acid with protamine sulfate, the natural type ectoine synthase is 
isolated and purified by subjecting it to salting-out with ammonium 
sulfate, a hydrophobic column chromatography, and a hydroxyapatite column 
chromatography. It has a molecular weight of about 19 kilodalton (kDa) 
measured by electrophoresis with SDS-polyacrylamide gel, and further, it 
has an isoelectric point of 4.2-4.4. It has also been determined that it 
has an amino acid sequence having 30 amino acid residues from the 
N-terminus as shown in SEQ ID NO: 3 according to an amino acid sequencer 
(Applied Biosystems). 
Moreover, the present inventors have succeeded to isolate a gene DNA coding 
for ectoine synthase from the gene DNA of Halomonas sp. KS-3 based on the 
above N-terminus amino acid sequence, and then determined the nucleotide 
sequence. The nucleotide sequence of the DNA coding for the ectoine 
synthase is shown in SEQ ID NO: 2. 
The DNA coding for the ectoine synthase can be isolated by the following 
procedure. That is, the 4.2 kbp-DNA-EcoRI-SalI fragment is cleaved with 
restriction endonucleases MboI and NspBII to isolate a fragment having 228 
base pairs of the nucleotide Nos. of from 3 to 230, and a fragment having 
178 base pairs of the nucleotide Nos. of from 231 to 408 in the nucleotide 
sequence as shown in SEQ ID NO: 2, and the both fragments are ligated by 
T4 DNA ligase to give a DNA fragment having 406 base pairs of the 
nucleotide Nos. of from 3 to 408. Thereafter, the truncated both termini 
thereof are repaired by a DNA chemically synthetized to give the desired 
DNA coding for ectoine synthase. Alternatively, the 4.2 kbp-DNA-EcoRI-SalI 
fragment is cleaved with restriction endonucleases AspAII (another name: 
BstEII) and NspBII to isolate a fragment of the nucleotide Nos. of from 46 
to 408, and then the truncated both termini thereof are repaired by a DNA 
chemically synthetized to give the desired DNA coding for ectoine 
synthase. 
The whole amino acid sequence of the ectoine synthase is determined based 
on the nucleotide sequence, which is as shown in SEQ ID NO: 1. It was 
confirmed that the sequence of N-terminus amino acids is completely 
identical with that of the 30 N-terminus amino acids of the natural type 
ectoine synthase. The ectoine synthase is a protein which is composed of 
137 amino acids and has a molecular weight of 15.5 kDa. The amino acid 
components are also well identical to the data determined (cf. Table 1) 
for the natural type ectoine synthase. The nucleotide sequence encoding 
the ectoine synthase as well as the corresponding amino acid sequence are 
shown in SEQ ID NO: 4. 
TABLE 1 
______________________________________ 
Amino acid components of ectoine synthase 
Nucleotide 
Amino acid Natural type enzyme 
sequence 
______________________________________ 
Lys 5 5 
His 8-9 9 
Trp .gtoreq.2 2 
Arg 7 7 
Asp/Asn 15-16 16 
Thr 9-10 10 
Ser 5 5 
Glu/Gln 19 18 
Pro 5 5 
Gly 11 11 
Ala 10 10 
Cys &gt;2 3 
Val 6-7 6 
Met 2 2 
Ile 8-9 9 
Leu 11 11 
Tyr 4 4 
Phe 4 4 
Total number &gt;133 137 
______________________________________ 
The columns of the natural type enzyme and the nucleotide sequence in the 
above Table 1 mean the amino acid components of the natural type ectoine 
synthase and the amino acid components of the ectoine synthase which is 
determined based on the nucleotide sequence, respectively. 
The present inventors have isolated the about 4.2 kbp DNA fragment obtained 
from Halomonas sp. KS-3 by cleaving by restriction endonucleases EcoRI and 
SalI. After confirming that the nucleotide sequence coding for the ectoine 
synthase as shown in SEQ ID NO: 2 is contained in this DNA fragment, it is 
inserted into plasmid pBR322 and introduced into E. coli, and then, it has 
been found that the transformed E. coli has the desired high osmotic 
tolerance so that it can grow even in a medium of a high salt 
concentration. Owing to the transformation the gene of enzyme effective 
for synthesis of ectoine exhibits its function, and thereby ectoine is 
synthetized and accumulated within the cells, as a result, E. coli has 
characteristics of high osmotic tolerance. 
According to the present invention, the DNA coding for an enzyme effective 
for synthesis of ectoine is recombined to a vector replicable within host 
cells, followed by introducing it into a host, and thereby, the host is 
given by a capability of synthetizing efficiently ectoine and hence 
acquires characteristics of high osmotic tolerance. By utilizing this 
technique, there can be prepared micro-organisms and plants which have 
high osmotic tolerance, and hence, it will be possible to develop an 
efficient fermentation method using said transformant, creation of plants 
having high resistance to drought, and improvement of soil in desert. 
The first object of the present invention is to provide a DNA coding for 
ectoine synthase. An example of a nucleotide sequence of the DNA coding 
for ectoine synthase is the sequence shown in SEQ ID NO: 2 and an allele 
thereof. The amino acid sequence of the ectoine synthase is shown in SEQ 
ID NO: 1. The amino acid sequence may partially be changed by 
modifications, deletions, additions, and the like in accordance with a 
genetic engineering technique. Such a modified enzyme is also included in 
the present invention as far as it has the essential enzymatic properties 
of ectoine synthase, and hence, the nucleotide sequence coding for the 
enzyme is also included in the present inventon. Based on the information 
of the amino acid sequence shown in SEQ ID NO: 1, a DNA coding for ectoine 
synthase can also be chemically synthetized by selecting the codons 
suitable for a host cell. 
The second object of the invention is to provide a DNA fragment containing 
a gene coding for ectoine synthase. Specifically, it is a DNA comprising 
about 4.2 kilobase pairs which is obtained from Halomonas sp. KS-3 by 
cleaving with restriction endonucleases EcoRI and SalI and has the 
nucleotide sequence as shown in SEQ ID NO: 2. 
The third object of the invention is to provide a recombinant DNA 
self-replicable within the host cells, which is prepared by recombining a 
DNA coding for ectoine synthase to a vector DNA self-replicable within the 
host cells. 
The fourth object of the invention is to provide a method for providing an 
ability of biosynthesis of ectoine to a host cell, which comprising 
introducing a recombinant DNA containing a DNA coding for ectoine synthase 
into a host cell. 
The fifth object of the invention is to provide microorganisms or plants 
which are transformed by introducing a recombinant DNA containing a DNA 
coding for ectoine synthase. 
The DNA coding for ectoine synthase of the invention can be detected by 
determining the nucleotide sequence of the product and then comparing it 
with the nucleotide sequence as shown in SEQ ID NO: 2, or by southern 
blotting hybridization using as a probe a fragment of the DNA having a 
nucleotide sequence as shown in SEQ ID NO: 2, or by transforming E. coli 
by introducing the gene and confirming the growth thereof in a medium 
having a high salt concentration. 
Determination of ectoine in cells: It is carried out by the steps of 
extracting the cells with a 10 times larger volume of a 70% ethanol at 
80.degree. C. for 10 minutes, filtering it with a glass filter to give a 
crude extract, removing ethanol from the crude extract by concentration 
under reduced pressure (at 35.degree. C.), adding an equivolume of 
chloroform to the concentrated solution, centrifuging (1,500 rpm, 10 
minutes), and again concentrating the supernatant under reduced pressure, 
diluting the concentrated solution with a distilled water, charging it 
onto a cation exchange column (DIAION SKLB, manufactured by Mitsubishi 
Chemical), washing it with water, eluting ectoine therefrom with 3N 
ammonium hydroxide, removing ammonium hydroxide from the eluate by 
concentrating under reduced pressure, and charging the resultant onto an 
anion exchange column (DIAION SAlOA, manufactured by Mitsubishi Chemical) 
to isolate the ectoine. The analysis of ectoine can be carried out by high 
performance liquid chromatography or a thin layer chromatography in 
accordance with a method of Peters, P., et al. (cf. FEMS Microbiol. Lett., 
Vol. 71, pp. 157-162, 1990) . 
Determination of the enzyme activity of ectoine synthase: It is carried out 
by adding an enzyme to be tested to a reaction mixture consisting of 80 mM 
Tris-HCl buffer (pH 9.5), 4.4 mM .alpha.-N-acetyl-diaminobutyric acid, 
0.77 M sodium chloride (the total volume of the reaction mixture: 45 
.mu.L), reacting at 15.degree. C. for 10 minutes, quenching the reaction 
by adding thereto an equiamount of 0.6% trifluoroacetic acid, and 
analyzing the amount of the produced ectoine in the reaction mixture by 
high performance liquid chromatography. One unit of the enzyme activity of 
ectoine synthase is defined as an amount of the enzyme which can produce 1 
.mu.mole of ectoine per one minute. 
In the present invention, the bacteria used for isolating the DNA coding 
for ectoine synthase is not limited to Halomonas sp. KS-3, but includes 
any microorganisms which are capable of producing ectoine. The known 
microorganisms are, for example, Ectothiorhodospira halochloris (American 
Type Culture Collection, accession number, ATCC 35916) (cf. Galinski, E. 
A. et al., Eur. J. Biochem., Vol. 149, pp. 135-139, 1985), Halomonas 
elongata (cf. Wohlfarth, A. et al., J. Gen. Microbiol., Vol. 136, pp. 
705-712, 1990), Vibrio costicola (cf. Regev. R. et al., Arch. Biochem. 
Biophys., Vol. 278, pp. 106-112, 1990). The DNA coding for an enzyme 
involved in the biosynthesis of ectoine can be isolated from a bacteria of 
the genus Halomonas or any other cells, which may have the same or highly 
homologous nucleotide sequence, by hybridization method using as a probe a 
DNA having the nucleotide sequence as shown in SEQ ID NO: 2. Moreover, 
based on the information of the nucleotide sequence as shown in SEQ ID NO: 
2 or of the amino acid sequence shown in SEQ ID NO: 1, a DNA coding for 
ectoine synthase can also be chemically synthetized by selecting the 
codons suitable for a host cell. Furthermore, a DNA coding for ectoine 
synthase which has a modified structure may also be produced by partially 
modifying the DNA coding for ectoine synthase in accordance with a 
site-specific mutation method (cf. Kunkel, T. A. et al., Methods in 
Enzymol., Vol. 154, pp. 367-392, 1987). 
The expression of a foreign gene within host cells can be done by the 
methods as described in many textbooks and literatures (for example, 
Molecular Cloning; A Laboratory Manual, 2nd Ed. Vol. 1-3, ed. by Sambrook, 
J. et al, Cold Spring Harbor Laboratory Press, New York 1989), and the 
basic theory thereof has already been established. A recombinant DNA being 
replicable and functioning in the host cells can be produced by adding a 
translation initiation codon at the upstream of a DNA coding for the 
desired protein to be expressed and a translation termination codon at the 
downstream thereof, adding regulatory genes such as a promoter sequence 
which can regulate the transcription (e.g. trp, lac, phoS, PL, SV40 early 
promoter), and inserting it into an appropriate vector (e.g. pHY300PLK, 
pBR322, pUCl9, pYAC-neo). The expression vector has preferably genetic 
information for replicating within host cells and can replicate therein 
and has preferably a gene to be functioned as a detectable marker. The 
most suitable vector is selected depending on the kinds of hosts. 
Moreover, a suitable promoter functionable within the host cells is also 
chosen. A regulatory gene derived from the host cells is preferable from 
the viewpoint of exhibiting the desired functions. 
Introduction of the gene into microorganisms (e.g. bacteria, yeast) and 
plants can be done by a calcium chloride method (e.g. Cohen, S. N. et al, 
Proc. Natl. Acad. Sci. USA, Vol. 69, pp. 2110-2114, 1972), a DEAE-dextran 
method (e. g. Current Protocols in Molecular Biology, Vol. 1, Chapter 9.2, 
ed. by Ausubel, F. M. et al, John Wiley & Sons, 1987), an electroporation 
method, a method using protoplast, a method using Ti plasmid, a method 
using a virus vector (e.g. Watson J. D. et al., Molecular Biology of 
Recombinant DNA, translated by Michio Matsuhashi et al., issued by 
Maruzen, 1993). 
The basic procedures of genetic engineering technique shall be referred to 
many literatures and technical texts, for example, Molecular Cloning; A 
Laboratory Manual, 2nd. Ed., Vol. 1-3, by Sambrook, J. et al, Cold Spring 
Harbor Laboratory Press, New York 1989; Current Protocols in Molecular 
Biology, Vol. 1-2, by Ausubel, F. M. et al, Current Protocols, 1993; and 
Watson J. D. et al., Molecular Biology of Recombinant DNA, translated by 
Michio Matsuhashi et al., issued by Maruzen, 1993. The various 
instruments, enzymes and agents used in the technical field of this 
invention are used in the manner along with each guidance, reference note 
and manual. 
The following abbreviations are used in order to simplify the description. 
A: Adenine 
C: Cytosine 
G: Guanine 
T: Thymine 
Ala: Alanine 
Arg: Arginine 
Asn: Asparagine 
Asp: Aspartic acid 
Cys: Cysteine 
Gln: Glutamine 
Glu: Glutamic acid 
Gly: Glycine 
His: Histidine 
Ile: Isoleucine 
Leu: Leucine 
Lys: Lysine 
Met: Methionine 
Phe: Phenylalanine 
Pro: Proline 
Ser: Serine 
Thr: Threonine 
Trp: Tryptophan 
Tyr: Tyrosine 
Val: Valine 
DNA: Deoxyribonucleic acid 
kDa: kilodalton 
EXAMPLES 
The present invention is illustrated in more detail by referring to the 
following examples, but it should not be construed to be limited thereto 
and the present invention includes any modification by a conventional 
technique in the technical field of this invention. 
Example 1 
Isolation of a gene DNA of a bacteria of the genus Halomonas: 
Halomonas sp. KS-3 was inoculated to a M63 medium (components: 1.4% 
KH.sub.2 PO.sub.4, 0.4% KOH, 0.2% (NH.sub.4).sub.2 SO.sub.4, 1 mM 
MgSO.sub.4, 3.9 .mu.M FeSO.sub.4, 0.4% glucose) and thereto were added 3% 
sodium chloride and 0.25% yeast extract, and the mixture was aerobically 
pre-cultured at 37.degree. C. overnight. This pre-culture broth was 
inoculated to a fresh M63 medium (100 ml) containing 3% sodium chloride in 
a concentration of 2% and it was aerobically cultured with shaking (140 
rpm) at 37.degree. C. After culturing for about 5 hours, when the culture 
broth had a turbidity of about 2 (absorbance at a wavelength of 660 nm), 
sodium chloride was added so as to be a final concentration of about 15%, 
and the mixture was further cultured for 5 hours. The cells were separated 
by centrifuge and washed to give cells (about 0.25 g). 
A DNA was isolated from the cells by a conventional method (cf. Current 
Protocols in Molecular Genetics, ed. by Ausubel F. M. et al., p. 431, Cold 
Spring Harbor Laboratory Press, New York, 1972). That is, the cells were 
lyzed with protease K, centrifuged (8,000 rpm, for 10 minutes) to remove 
the residues, and then treated with ribonuclease. Thereafter, protein was 
removed by extracting with a mixture of an equiamount of 
phenol/chloroform, and then subjected to precipitation with ethanol to 
give the desired gene DNA of Halomonas bacteria (about 0.8 mg). 
Example 2 
Isolation of a DNA coding for ectoine synthase: 
There were chemically synthetized two kinds of DNAs having 25 nucleotides 
which coded the amino acid sequences corresponding to both termini of the 
N-terminus amino acid sequences of the ectoine synthase as shown in SEQ ID 
NO: 3. The nucleotide sequences had the following formulae I! and II!, 
respectively. 
EQU 5'-TGATHGTNMGNAAYYTNGARGARGC-3' I! 
EQU 5'-GNTYNSWNTYNGMNCANSWYAGGGT-3' II! 
In the above formulae I! and II!, M is either A or C, R is either A or G, 
W is either A or T, S is either G or C, Y is either C or T, H is either A 
or C or T, N is either A or G or C or T. 
By using the chemically synthetized DNAs having the above formulae I! and 
II! as a primer, and also using the gene DNA of Halomonas bacteria 
obtained in Example 1 as a template, the desired gene DNA was specifically 
amplified by polymerase chain reaction with Ampli Taq Polymerase 
(manufactured by Perkin-Elmer Company) in accordance with the 
specification attached thereto. The temperature for modification, 
annealing and polymerase reaction were 95.degree. C., 45.degree. C., and 
72.degree. C., respectively. 
The reaction product was subjected to an electrophoresis with a low melting 
agarose gel and then extraction to isolate the desired DNA fragment having 
90 base pairs. This DNA fragment was inserted into plasmid pUC19 at the 
cleavage site with a restriction enzyme SmaI. The recombinant plasmid was 
introduced into E. coli DH5.alpha.F' (manufactured by Life Technologis, 
Inc.) to prepare a transformant. A recombinant plasmid was recovered from 
the transformant thus obtained, and the nucleotide sequence of the 
recombined DNA fragment having 90 base pairs was determined by dideoxy 
method. The amino acid sequence coded by the nucleotide sequence was 
completely identical to the sequence shown in SEQ ID NO: 3, by which it 
was confirmed to be the desired DNA fragment. 
Example 3 
Isolation of the gene DNA containing a DNA coding for ectoine synthase: 
The gene DNA of Halomonas bacteria obtained in Example 1 was cleaved by 8 
kinds of restriction endonucleases as mentioned below, and the fragments 
were isolated by an electrophoresis using an agarose gel. The used 
restriction endonucleases were EcoRI, SalI, ScaI, KpnI, BanHI, PvuII, 
NruI, and PstI. After isolating by the electrophoresis using an agarose 
gel, each DNA fragment was subjected to detection by southern blotting 
hybridization using as a probe a DNA fragement coding for ectoine 
synthase. As a result, a DNA containing completely the DNA coding for 
ectoine synthase, i.e. a DNA of about 4.2 kbp in size cleaved with EcoRI 
and SalI (this fragment is designated as "4.2 kbp-DNA-EcoRI-SalI 
fragment") was isolated. The result of analysis of the gene DNA of 
Halomonas bacteria with restriction endonucleases is shown in the 
accompanying FIG. 1. 
Separately, there was isolated a large DNA fragment containing replication 
origin, ampicillin resistant gene, etc., which was obtained by cleaving 
plasmid pBR322 with EcoRI and SalI, (this fragment was designated as 
"pBR322-EcoRI-SalI fragment"). The 4.2 kbp-DNA-EcoRI-SalI fragment and the 
pBR322-EcoRI-SalI fragment were ligated with T4 DNA ligase to prepare a 
cyclic recombinant plasmid DNA. This recombinant DNA was designated as 
"pECT101". 
Example 4 
Nucleotide sequence of a DNA coding for ectoine synthase: 
For the purpose of determining the nucleotide sequence of the 4.2 
kbp-DNA-EcoRI-SalI fragment prepared in Example 3, it was inserted into 
the cloning region of a vector pBluescript II SK+ (manufactured by Toyobo 
Co., Ltd.) (this was designated as "pECT201"), and the nucleotide sequence 
was determined by using pBluescript II Exo/Mung DNA Sequencing System 
(manufactured by Stratagene Inc.). The nucleotide sequence coding for 
ectoine synthase and whole amino acid sequence are shown in SEQ ID NOS: 4 
and 5. 
EXAMPLE 5 
Transformation with the DNA coding for ectoine synthase: 
The recombinant DNA (pECT101) obtained in Example 3, which has been 
recombined with the 4.2 kbp-DNA-EcoRI-SalI fragment containing completely 
the DNA coding for ectoine synthase, was introduced into E. coli 
DH5.alpha.F' in accordance with the method described in Molecular Cloning; 
A Laboratory Manual, 2nd Ed., Vol. 1, 1.77-1.81 (ed. by Sambrook, J. et 
al., Cold Spring Harbor Laboratory Press, New York, 1989) to give a 
transformant having ampicillin resistance (it was designated as "E. coli 
DH5/pECT101"). Moreover, the transformant was subjected to hybridization, 
by which it was confirmed that the transformant contained a DNA coding for 
ectoine synthase. 
The transformant E. coli DH5/pECT101 was inoculated to a M63 medium 
incorporated with 0.02% casamic acid and 2 .mu.g/ml thyamine, and cultured 
at 37.degree. C. When the turbidity of the culture broth (the absorbance 
at a wavelength of 610 nm) became about 0.2, sodium chloride was added so 
as to be a final concentration of 5%, and the culture was continued, where 
the growth state of the bacteria was observed with Bioscanner OT-BS-48 
(manufactured by Ohtake Seisakusho). Plasmid pBR322 was introduced into E. 
coli DH5.alpha.F' likewise to give a transformant having ampicillin 
resistance (it was designated as "E. coli DH5/pBR322"), which was used as 
a reference transformant. As a result, as is shown in the accompanying 
FIG. 2, the reference transformant (E. coli DH5/pBR322) discontinued to 
grow in a medium having a high salt concentration. On the contrary, the 
transformant E. coli DH5/pECT101 grew well even in a medium having a high 
salt concentration. This means that the product of the invention acquired 
such characteristics as being capable of growing even under the 
environmental condition of a high salt concentration as well as the high 
osmotic pressure environment induced by the high salt concentration 
because of giving an ability of synthesis of ectoine by the introduction 
of a gene of ectoine synthase, and thereby having high tolerance to such 
environmental conditions. 
UTILIZATION IN INDUSTRIES 
By a genetic engineering technology using the gene DNA of ectoine synthase 
of the present invention, there can be produced ectoine which may be 
useful as a water-retaining material. Besides, when the DNA of the present 
invention is recombined into various microorganisms and plants by using an 
appropriate expression vector, there can be expressed a product having 
characteristics of being tolerant even under environmental conditions of a 
high osmotic pressure, and thereby, the process for cultivation of 
microorganisms may be improved so as to be able to proceed even in a high 
concentration and further there can be obtained plants having high 
resistance to drought and also tolerance to high osmotic pressure. This 
will be effective for revival of micro-organisms in soil of desert, and 
further creation of plants which can grow even in a droughty ground or at 
a seaside. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 5 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 137 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: Not R - #elevant 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- Met Ile Val Arg Asn Leu Glu Glu Ala Arg Gl - #n Thr Asp Arg Leu Val 
# 15 
- Thr Ala Glu Asn Gly Asn Trp Asp Ser Thr Ar - #g Leu Ser Leu Ala Glu 
# 30 
- Asp Gly Gly Asn Cys Ser Phe His Ile Thr Ar - #g Ile Phe Glu Gly Thr 
# 45 
- Glu Thr His Ile His Tyr Lys His His Phe Gl - #u Ala Val Tyr Cys Ile 
# 60 
- Glu Gly Glu Gly Glu Val Glu Thr Leu Ala As - #p Gly Lys Ile Trp Pro 
#80 
- Ile Lys Pro Gly Asp Ile Tyr Ile Leu Asp Gl - #n His Asp Glu His Leu 
# 95 
- Leu Arg Ala Ser Lys Thr Met His Leu Ala Cy - #s Val Phe Thr Pro Gly 
# 110 
- Leu Thr Gly Asn Glu Val His Arg Glu Asp Gl - #y Ser Tyr Ala Pro Ala 
# 125 
- Asp Glu Ala Asp Asp Gln Lys Pro Leu 
# 135 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 411 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- ATGATCGTTC GCAATCTCGA AGAAGCGCGC CAGACCGACC GTCTGGTCAC CG - #CCGAAAAC 
60 
- GGCAACTGGG ACAGCACCCG CCTGTCGCTG GCCGAAGATG GTGGCAACTG CT - #CCTTCCAC 
120 
- ATCACCCGCA TCTTCGAGGG TACCGAGACC CACATCCACT ACAAGCATCA CT - #TCGAGGCT 
180 
- GTTTATTGCA TCGAAGGCGA GGGCGAAGTG GAAACCCTGG CCGATGGCAA GA - #TCTGGCCC 
240 
- ATCAAGCCGG GTGACATCTA CATCCTCGAC CAGCACGACG AGCACCTGCT GC - #GCGCCAGC 
300 
- AAGACCATGC ACCTGGCCTG CGTGTTCACG CCGGGCCTGA CCGGCAACGA AG - #TGCACCGC 
360 
# 411ACGCACC TGCCGACGAA GCCGACGACC AGAAGCCGCT G 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 30 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: Not R - #elevant 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- Met Ile Val Arg Asn Leu Glu Glu Ala Arg Gl - #n Thr Asp Arg Leu Val 
# 15 
- Thr Ala Glu Asn Gly Asn Trp Asp Ser Thr Ar - #g Leu Ser Leu 
# 30 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 414 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA to mRNA 
- (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..411 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- ATG ATC GTT CGC AAT CTC GAA GAA GCG CGC CA - #G ACC GAC CGT CTG GTC 
48 
Met Ile Val Arg Asn Leu Glu Glu Ala Arg Gl - #n Thr Asp Arg Leu Val 
# 15 
- ACC GCC GAA AAC GGC AAC TGG GAC AGC ACC CG - #C CTG TCG CTG GCC GAA 
96 
Thr Ala Glu Asn Gly Asn Trp Asp Ser Thr Ar - #g Leu Ser Leu Ala Glu 
# 30 
- GAT GGT GGC AAC TGC TCC TTC CAC ATC ACC CG - #C ATC TTC GAG GGT ACC 
144 
Asp Gly Gly Asn Cys Ser Phe His Ile Thr Ar - #g Ile Phe Glu Gly Thr 
# 45 
- GAG ACC CAC ATC CAC TAC AAG CAT CAC TTC GA - #G GCT GTT TAT TGC ATC 
192 
Glu Thr His Ile His Tyr Lys His His Phe Gl - #u Ala Val Tyr Cys Ile 
# 60 
- GAA GGC GAG GGC GAA GTG GAA ACC CTG GCC GA - #T GGC AAG ATC TGG CCC 
240 
Glu Gly Glu Gly Glu Val Glu Thr Leu Ala As - #p Gly Lys Ile Trp Pro 
# 80 
- ATC AAG CCG GGT GAC ATC TAC ATC CTC GAC CA - #G CAC GAC GAG CAC CTG 
288 
Ile Lys Pro Gly Asp Ile Tyr Ile Leu Asp Gl - #n His Asp Glu His Leu 
# 95 
- CTG CGC GCC AGC AAG ACC ATG CAC CTG GCC TG - #C GTG TTC ACG CCG GGC 
336 
Leu Arg Ala Ser Lys Thr Met His Leu Ala Cy - #s Val Phe Thr Pro Gly 
# 110 
- CTG ACC GGC AAC GAA GTG CAC CGC GAA GAC GG - #T TCC TAC GCA CCT GCC 
384 
Leu Thr Gly Asn Glu Val His Arg Glu Asp Gl - #y Ser Tyr Ala Pro Ala 
# 125 
# 414 AC CAG AAG CCG CTG TAA 
Asp Glu Ala Asp Asp Gln Lys Pro Leu 
# 135 
- (2) INFORMATION FOR SEQ ID NO:5: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 137 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- Met Ile Val Arg Asn Leu Glu Glu Ala Arg Gl - #n Thr Asp Arg Leu Val 
# 15 
- Thr Ala Glu Asn Gly Asn Trp Asp Ser Thr Ar - #g Leu Ser Leu Ala Glu 
# 30 
- Asp Gly Gly Asn Cys Ser Phe His Ile Thr Ar - #g Ile Phe Glu Gly Thr 
# 45 
- Glu Thr His Ile His Tyr Lys His His Phe Gl - #u Ala Val Tyr Cys Ile 
# 60 
- Glu Gly Glu Gly Glu Val Glu Thr Leu Ala As - #p Gly Lys Ile Trp Pro 
# 80 
- Ile Lys Pro Gly Asp Ile Tyr Ile Leu Asp Gl - #n His Asp Glu His Leu 
# 95 
- Leu Arg Ala Ser Lys Thr Met His Leu Ala Cy - #s Val Phe Thr Pro Gly 
# 110 
- Leu Thr Gly Asn Glu Val His Arg Glu Asp Gl - #y Ser Tyr Ala Pro Ala 
# 125 
- Asp Glu Ala Asp Asp Gln Lys Pro Leu 
# 135 
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