JPH09308486A - Gene capable of coding amino acid sequence causing enzymatic activities of xylose metabolic system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new subject gene capable of coding a xylose metabolic system enzyme such as a xylulokinase, etc., having a specific amino acid sequence, facilitating a xylose fermentation by all kinds of Tetragenococcus harophilus and useful for brewing of a soy source. SOLUTION: This gene is a new one capable of coding an amino acid sequence causing enzymatic activities of a xylose metabolic system comprising a xylulokinase substantially having an amino acid sequence of formula I and/or a xylose porter substantially having an amino acid sequence of formula II, facilitates a xylose fermentation by all kinds of Tetragenococcus harophilus and is extremely useful for a brewing of a soy source, a fermentation of cereals, a pentose fermentation used for the production of a biomass, etc. The gene is obtained by isolating the chromosomal DNA from Tetragenococcus harophilus I strain (FERM BP-1302) by a conventional method, preparing a library and further screening by a probe comprising a part of xylose metabolic system gene.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to Tetragenococcus [Pediococcu].
s)]-A gene encoding an amino acid sequence which brings about the enzyme activity of xylose-metabolizing system derived from halophilus.

[0002]

2. Description of the Related Art Conventionally, xylulokinase (EC 2.7.1.17) and / or xylose transporter, which is a xylose-metabolizing enzyme derived from Tetragenococcus halophilus, is
It is a protein whose structure has not been purified. In particular, the xylose transporter is a membrane protein and is therefore extremely difficult to purify, and no protein or gene has been identified in Gram-positive bacteria. Therefore, the exact amino acid sequences of these enzymes have not yet been elucidated.

On the other hand, the fermentation of xylose is a useful fermentation applied to the brewing of soy sauce, etc. However, until now, they have only been made by a special mutant strain of a microorganism (Japanese Patent Laid-Open No. 5-58). 49440, JP-A-5-49441, etc.). By transforming these xylose-metabolizing genes into tetragenococcus, xylose-metabolizing ability can be imparted to any strain, and the barrier limited to the above-mentioned special mutant strain is overcome.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to clone a gene for xylose metabolism enzyme and provide the gene. If this gene can be expressed in a host microorganism, xylose can be fermented in any Tetragenococcus halophilus.

[0005]

[Means for Solving the Problems] The inventors of the present invention have found that for a phage library of tetragenococcus halophilus chromosomal genes, the amino acid sequence constituting the xylose isomerase active center of the genus Bacillus [Wi
lhelm M, Hollenberg CP, Nucleic Acid Research 15: 5
717-5722, (1985)], homology screening (by plaque hybridization method) was performed using an oligonucleotide probe prepared on the basis of the nucleotide sequence deduced from the above. As a result, the xylose-metabolizing enzyme gene derived from Tetragenococcus halophilus was successfully isolated, and its peptide structure was successfully determined, thus completing the present invention.

That is, the present invention provides a xylose-metabolizing enzyme activity comprising a xylulokinase having substantially the amino acid sequence shown in SEQ ID NO: 1 and / or a xylose transporter having substantially the amino acid sequence shown in SEQ ID NO: 2. Is a gene encoding an amino acid sequence that produces A specific example of this gene is the DNA sequence shown in SEQ ID NO: 3. The term "substantially" in "substantially the xylulokinase having the amino acid sequence shown in SEQ ID NO: 1" means not only the xylulokinase having the amino acid sequence shown in SEQ ID NO: 1 but also the above. It is meant that one or more amino acids are added, deleted or substituted in the amino acid sequence, and that it also includes an amino acid sequence that causes xylulokinase activity. Also,
“Substantially” in “substantially xylose transporter having the amino acid sequence shown in SEQ ID NO: 2” means the same as above.

Hereinafter, the present invention will be described in detail. The amino acid sequence of the xylose isomerase active center of the genus Bacillus [Sequence 1 (Trp Gly Gly Arg Glu Gly) and Sequence 2 (Ile G
lu Pro Lys Pro Lys Glu Pro)], the oligonucleotide 1 (TGG G corresponding to Sequence 1 based on the DNA sequence predicted from
GT / C GGT / C GAA / G GG) and oligonucleotide 2 corresponding to sequence 2 (GGT / C TCI TT / GI GGT / C TTI GGT / C TCT / G / A
AT) (where I represents inosine) mixed probe was synthesized by a DNA synthesizer manufactured by Applied Biosystems (ABI).

Any donor microorganism having a xylose-metabolizing enzyme active substance may be used as long as it belongs to the genus Tetragenococcus and produces a xylose-metabolizing enzyme (xylulokinase and / or xylose transporter). For example, Tetragenococcus halophilus I (JP-A-63-219368) and the like can be mentioned. For culturing this microorganism, for example, the medium and culture method described in JP-A-5-49440 are used. The culture obtained by culturing this microorganism is centrifuged, for example, at 10,000 rpm for 10 minutes to obtain cells of Tetragenococcus halophilus I. Chromosomal DNA is obtained from the cells by a known method [eg, the method described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Vol. 1, No. 2 (1987)].
Then, a restriction enzyme such as Sau III AI is added to this chromosomal DNA.
Temperature (Takara Shuzo) or HinD III (Takara Shuzo)
30 ℃ or more, preferably 37 ℃, enzyme concentration 1-10 units / m
Digested with l for 10 to 20 hours, various chromosome D
Obtain NA fragment. This chromosomal DNA fragment was electrophoresed on a 0.7% agar plate by the method described in Current Protocols in Molecular Biology Vol. 1, No. 2 etc., and a fragment having a molecular weight of 4 to 20 kb (kilobase) was quantified.
It is recovered from an agar plate and purified by a Quiaquick Gel Extraction Kit manufactured by gen.

On the other hand, the vector that can be used in the present invention may be any vector as long as it can be used, and examples thereof include a plasmid vector and a phage vector. Preferred examples thereof include phage λEMBL3 (produced by Promega). ) Or λZap Express (manufactured by Stratagene). And, for example, λEMB
L3 vector DNA with BamHI and EcoRI, λZap Expr
Each ess was HinDIII at temperature 37 ℃, enzyme concentration 5-10
The digested vector fragment is obtained by digesting with 0 unit / ml for 1 hour or more, preferably 1 to 3 hours. Furthermore, if necessary, these vector fragments are purified by a known method such as phenol extraction treatment, phenol-chloroform extraction treatment, or ethanol precipitation treatment.

Then, the above-mentioned chromosomal DNA fragment mixture derived from Tetragenococcus halophilus I and containing a xylose metabolism gene is mixed with the above vector fragment, for example, 4 to 16 units of T4-DNA ligase at 1 to 10 units / ml.
Reactant phage DNA at 12 ℃ for 16 to 16 hours
Get. Recombinant phage obtained in this way
For DNA, by conventional in vitro-packaging methods, phage particles can be formed, and the phage can obtain recombinant phage plaques carrying various DNA fragments. E. coli P2 392 (Toyobo) (for λEMBL3) or E. coli XLOLR (for Stratagene) (for λZap Express)
Plaques of recombinant phages carrying various DNA fragments can be obtained. DNA containing xylose metabolism genes from plaques thus obtained
Search for recombinant phages carrying the fragment [γ-32P]
Plaque hybridization using the above oligonucleotides 1 and 2 as probes, labeled at the 5'end with ATP (manufactured by Amersham Japan) [Current Protocols in Molecular Biology, Vol. 1, No. 6 (1987)]. Method].

From this recombinant phage (containing a DNA fragment containing the xylose metabolism gene therein),
Known methods (for example, molecular cloning (Mol
ecular Cloning, pp. 371-372, Cold Spring Harbor Laboratory
r Laboratory) (1982)) to obtain purified phage DNA.

The recombinant λEMBL3 phage DNA purified as described above is digested with the restriction enzyme SalI and subjected to agarose gel electrophoresis. For example, the DNA determined to contain the xylose metabolism gene as described above. Insert only the Quiagen Quiaquick Gel Extrac
Purification using Kion Quick Gel Extraction Kit gives a purified Sal I fragment (about 4KB). This fragment is ligated to a plasmid vector such as pBluescript II SK or KS (+/-) (manufactured by Stratagene). These plasmid vectors were digested with Sal I (pBluescript II) (temperature: 37 ° C, enzyme concentration: 5-100 units / ml
For more than an hour, preferably 1 to 3 hours), the cleaved vector DNA obtained here and the purified Sal I fragment are reacted with T4-DNA ligase to obtain a recombinant DNA.

A DN containing xylose metabolism system genes
The recombinant λZap Express phage having the A fragment was recovered in the form of containing a fragment of about 4.6 kb cloned by the Stratagene Rapid Excision Kit in the plasmid pBK-CMVX. The 4.6 kb HinD III insert fragment contained in this plasmid pBK-CMVX is also a DNA fragment containing a gene encoding the xylose metabolic system.

Escherichia coli JM109 (manufactured by Takara Shuzo Co., Ltd.) was transformed with this recombinant DNA to obtain a strain containing a gene for xylose metabolism, and then a recombinant purified from this strain.
Obtain DNA. The transformation and the acquisition of recombinant DNA were performed by the method described in Current Protocols in Molecular Biology, Vol. 1, No. 1 (1987). Using DNA containing this xylose metabolism system gene,
The base sequence of the xylose metabolism system gene is analyzed (see SEQ ID NO: 3) to determine the amino acid sequence of the translated polypeptide (see SEQ ID NOs: 1 and 2). The gene encoding the amino acid sequence thus determined is the gene for the xylose metabolism enzyme activator polypeptide.

It should be noted that in the amino acid sequence shown in SEQ ID NO: 1 or 2, one or more amino acids are added, deleted or substituted, respectively, and the polyamino acid sequence has xylulokinase or xylose transporter activity. All genes encoding peptides are included in the present invention. Then, any method may be used to obtain these genes, for example, a method of treating the gene with a mutagen to generate a point mutation or a deletion mutation; selectively cleaving the gene, and then selecting a selected nucleotide. And a method of ligating the gene; oligonucleotide mutagenesis and the like.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described more specifically with reference to Examples. However, the technical scope of the present invention is not limited by these examples.

[0017]

【Example】

(1) Preparation of Tetragenococcus halophilus I chromosomal DNA 1 Tetragenococcus (Pediococcus) halofilus I strain (FERM BP-1302) was added to DIFCO containing 5% (W / V) NaCl.
1 L of MRS medium manufactured by the company was inoculated and statically cultured at a temperature of 30 ° C. for 72 hours to obtain a culture. This culture at 10,000 rpm 1
After centrifuging for 0 minutes by a conventional method to obtain 4 g of wet cells, a known method [Current Protocols in Molecular Biology, Vol. 1, No. 2 (1987) was used.
Chronograph), and chromosomal DNA was isolated. Next, 50 μg of this chromosomal DNA and 50 units of restriction enzymes Sau III AI or HinD III (both manufactured by Takara Shuzo Co., Ltd.)
Tris buffer [containing 100 mM NaCl, 10 mM MgSO ₄ , 1 mM dithiothreitol (pH 7.5)] and 10 mM Tris buffer [5
Contains 0 mM NaCl, 10 mM MgSO ₄ , 1 mM dithiothreitol (pH
7.5)] and reacted at 37 ° C. for 30 minutes (Sau III AI) or 16 hours (HinD III). DNA of about 4 to 20 kb size was obtained by subjecting the reaction-completed solution to electrophoresis of 0.7% (W / V) agarose gel (manufactured by Takara Shuzo) by a conventional method.
The fragments are transferred to the Quiagen Quiaquick Gel Extractio.
n Kit (Kia quick gel extraction kit) was used for purification. These digested fragments include a DNA fragment containing a gene encoding the xylose metabolic system.

Purified Sau III AI fragment and HinD III
Fragment 1 unit of alkaline phosphatase
(Takara Shuzo Co., Ltd.) and 50 mM Tris-hydrochloric acid buffer (pH 8) were added, and the mixture was reacted at a temperature of 37 ° C. for 45 minutes to remove the phosphate at the 5 ′ end. The reaction-terminated solution was subjected to a phenol extraction treatment by a conventional method, and then subjected to an ethanol precipitation treatment to obtain tetragenococcus halophilus I chromosome DNA.
Sau III AI and HinD III digestion fragment mixture (4-20K
b) (This includes a DNA fragment containing a gene encoding the xylose metabolism system.) 5 μg was obtained.

(2) Preparation of recombinant phage λXYL 2 μg of λEMBL3 phage DNA (manufactured by Promega) and 10 units each of 10 restriction enzymes Bam HI and Eco RI (manufactured by Takara Shuzo)
mM Tris buffer [50 mM NaCl, 10 mM MgSO ₄ , containing 1 mM dithiothreitol (pH 7.5)] was mixed and reacted at 37 ° C. for 2 hours, then 15 mM ethylenediaminetetraacetic acid (EDTA) (pH 8) was added to 70 ° C. The treatment was carried out for 10 minutes to stop the enzymatic reaction.

The reaction-terminated solution was subjected to phenol extraction treatment by a conventional method, and then isopropanol 1 precipitation treatment to remove small DNA fragments, followed by ethanol precipitation treatment to obtain Sau I.
ΛEMBL3 phage DNA capable of incorporating IIAI fragment
I got

2 μg of λZap Express phage DNA (manufactured by Stratagene) and restriction enzyme HinD III (manufactured by Takara Shuzo) 10
The unit was replaced with 50 mM Tris buffer [100 mM NaCl, 10 mM MgSO.
₄ , containing 1 mM dithiothreitol (pH 7.5)],
After reacting at 37 ° C for 2 hours, 15 mM ethylenediaminetetraacetic acid (E
DTA) (pH 8) was added and treated at 70 ° C. for 10 minutes to stop the enzymatic reaction. The reaction-terminated solution was subjected to a phenol extraction treatment suitable for a conventional method and ethanol precipitation treatment to obtain λZap Express phage DNA capable of incorporating the HinD III fragment.

Then, 1 μg of λEMBL3 phage DNA into which this Sau III AI fragment can be incorporated, and the Sau III AI-digested tetragenococcus.
Halophilus I chromosomal DNA fragment mixture (4-20k
b) 3 μg and 1 unit of T4 DNA ligase (Takara Shuzo) were added to 6.6 mM MgCl2, 10 mM dithiothreitol and 1
It was added to 66 mM Tris buffer (pH 7.5) containing 0 mM ATP and reacted at a temperature of 16 ° C. for 16 hours to ligate the DNA. Then, 1 μg of this recombinant phage DNA was subjected to a usual in vitro packaging method, for example, Gigapack III Gold Packing Extract manufactured by Stratagene.
First, it is incorporated into phage particles and infected with Escherichia coli as phages, chloroform is added to lyse the cells, and 50 mM Tris-HCl buffer (100 mM NaCl, 10 mM MgSO 4 is added).
₄ , containing 0.01% (W / V) gelatin). Similarly Hin
ΛZap Express phage DNA capable of incorporating D III fragment
1 μg, 3 μg of a mixture of chromosomal DNA fragments of Pediococcus halophilus I (4 to 20 kb) digested with HinD III obtained in the above item (1), and 1 unit of T4 DNA
Ligase (Takara Shuzo) was added to 66 mM Tris buffer (pH 7.5) containing 6.6 mM MgCl ₂ , 10 mM dithiothreitol and 10 mM ATP, and reacted at a temperature of 16 ° C. for 16 hours,
The DNA was ligated.

Next, 1 μm of this recombinant phage DNA
g is a standard in vitro packaging method, for example, Gigapack III Gold Packaging Extrac manufactured by Stratagene.
First, it was incorporated into phage particles by t, and after infecting Escherichia coli as a phage, chloroform was added to lyse the cells, and 50 mM Tris-HCl buffer [100 mM NaCl, 10 mM was added.
mM MgSO ₄ , containing 0.01% (W / V) gelatin].

(3) Preparation of probe for search From the recombinant phages thus obtained, Sau III AI fragment containing the xylose metabolism gene or Hi
In order to search for recombinant phages carrying the nD III fragment, the above oligo DNA was labeled with the current protocol.
The 5'end was labeled with [γ-32P] ATP (manufactured by Amersham Japan) by the method described in Molecular Biology, Volume 1, No. 6 (1987).

(4) Separation of Phage DNA Carrying Xylose Metabolism Gene Using the above-mentioned probe, a known method [Current Protocols in Molecular Biology, Vol. 1,
No. 6 (1987)]. The phage having DNA hybridized with this probe was used as recombinant phage DNAs λEMBL-Xyl and λZ having xylose metabolism gene.
Obtained as ap-Xyl.

(5) Recombinant phage DNA, λEMBL-Xy
Isolation of l and λZap-Xyl.

Escherichia coli LE392 (manufactured by Promega) 1 m of culture solution
1 was added with 0.1% (W / V) glucose and 8 mM MgSO ₄
-Y medium [1% (W / V) bactotryptone, 0.5% (W / V) bacto yeast extract, 0.5% NaCl (pH7.2)] 30 ml was inoculated and prepared in item (4). Lysate by plating of recombinant phage λEMBL-Xyl containing xylose metabolism genes (including 2.0 to 2.5 x 109 phages)
Was cultivated with shaking at a temperature of 37 ° C. until the LE392 was lysed (3 to 5 hours), 0.3 ml of chloroform was further added, and the mixture was shaken for 15 minutes. 15 times this lysate at 8,000 rpm
After centrifugation for 30 minutes, 30 μg of RNase was added to the supernatant, and after reacting at 37 ° C. for 30 minutes, 20% polyethylene glycol (PEG) /2.5 M NaCl equivalent to the lysate was added, PEG precipitation was performed at 0 ° C. for 1 hour. This precipitate was added to 50 mM Tris-HCl buffer [100 mM NaCl, 10 mM MgSO ₄ ,
0.1% (W / V) containing gelatin] and subjected to phenol extraction by a conventional method, followed by isopropanol precipitation treatment and ethanol precipitation treatment to obtain recombinant phage λEMBL-Xyl.
I got the DNA. Similarly, recombinant phage λZap-Xyl DNA was obtained using Escherichia coli XLOLR strain (manufactured by Stratagene).

(6) Preparation of recombinant plasmids pBKS + 2-2 and pBK-CMVX 2.5 μg of plasmid pBluescriptII KS + DNA (Stratagene) and restriction enzyme, Sal I 10 units in 50 mM Tris buffer [100 mM NaCl, 10 mM] MgSO ₄ , containing 1 mM dithiothreitol (pH 7.5)] and reacted at a temperature of 37 ° C. for 4 hours to obtain a digestive solution. The solution was subjected to phenol extraction and ethanol precipitation by a conventional method, and then 1 unit of alkaline It was added together with phosphatase (manufactured by Takara Shuzo Co., Ltd.) to 50 mM Tris-HCl buffer (pH 8) and reacted at a temperature of 37 ° C. for 45 minutes to remove the phosphate at the 5 ′ end. This reaction-terminated solution was subjected to a phenol extraction treatment and an ethanol precipitation treatment by a conventional method to obtain a plasmid pBluescriptII KS + DNA digested with SalI and HinDIII.

Then, 5 μg of the recombinant phage λEMBL-XylDNA obtained in the above item (5) and the restriction enzyme Sal
10 units of I (Takara Shuzo) 50 mM Tris buffer
[Containing 100 mM NaCl, 10 mM MgSO ₄ , 1 mM dithiothreitol
(pH 7.5)] and reacted at a temperature of 37 ° C. for 16 hours to obtain a digestive juice. The digested solution was electrophoresed on 0.7% (W / V) agarose gel, and only the Sal I fragment of about 4 kb was quantified by Quiagen.
(Qiagen) Quiaquick Gel Extraction Kit (Qiaquick Gel Extraction Kit)
0.1 μg of I fragment was obtained. The fragment is a DNA fragment containing a gene encoding the xylose metabolism system.

The Sal I fragment thus obtained, 0.1 μg, S
Plasmid pBluescript II KS + DNA0.1 digested with al I
μg and 1 unit of T4 DNA ligase (Takara Shuzo) were added to 6.6 mM MgCl2, 10 mM dithiothreitol and 10 mM.
It was added to 66 mM Tris buffer (pH 7.5) containing ATP and reacted at a temperature of 16 ° C. for 16 hours to ligate the DNA. Then, the ligated DNA was used to transform Escherichia coli JM109 (manufactured by Takara Shuzo Co., Ltd.) by a known method [a method described in Current Protocols In Molecular Biology, Volume 1, No. 3 (1987)]. The transformed strain
g / ml ampicillin, 0.5 mM isopropyl-β-thiogalactoside (IPTG) and 0.004% (W / V) 5-bromo-4-chloro-3-
T containing indolyl-β-D-galactoside (X-GAL), respectively
-Y agar plate medium for 24 hours at 37 ℃,
Those forming a white colony contained the xylose metabolism system gene, and pBKS + 2-2 was obtained as one of them.

Recombinant phage λZap-Xyl was recovered in the form of containing in the plasmid pBK-CMVX a fragment of about 4.6 kb cloned by the Stratagene Rapid Excision Kit. This plasmid
The 4.6 kb HinD III insert fragment contained in pBK-CMVX is also a DNA fragment containing a gene encoding the xylose metabolic system.

(7) Isolation of recombinant plasmid E. coli JM10 was added to TY medium containing 50 μg / ml of ampicillin.
Inoculate 9 (pBKS + 2-2) or E. coli JM109 (pBK-CMVX),
Shaking culture was performed at a temperature of 37 ° C. for 20 to 24 hours to obtain a culture solution. Next, from this culture solution, Current Protocols
In Molecular Biology, Volume 1, Issue 6 (1
(987) and the plasmid was extracted by the alkaline method described in (1987), and 100 μg of the plasmid was obtained using a Qiagen-maxi-plasmid purification kit manufactured by Qiagen.

(8) Contains xylose metabolism system genes
Analysis of DNA base sequence Plasmids pBKS + 2-2 and pBK- obtained in item (7) above
Regarding the insert fragment incorporated into CMVX, using the Stratogene ExoIII / Mung Bean DNA deletion kit, for those fragments having various lengths were prepared, diprimer with double-stranded DNA-tuck-sequencing-
Kit (manufactured by Applied Biosystems) and 50% (W /
V) urea containing 6% (W / V) polyacrylamide gel (Nati
Onal Diagnostics was used to analyze the nucleotide sequence with a DNA sequencer 370A (Applied Biosystems).

The base sequences of the obtained xylulokinase and xylose transporter are shown in SEQ ID NO: 3. The amino acid sequence of the xylulokinase polypeptide translated by the gene is shown in SEQ ID NO: 1, and the amino acid sequence of the xylulokinase polypeptide translated by the xylose transporter is shown in SEQ ID NO: 2, respectively. The xylulokinase of SEQ ID NO: 1 has a sequence of SEQ ID NO: 3 from 17 to 1522 bp, SEQ ID NO: 2
Xylose transporter of SEQ ID NO: 3 from 1842 to 32
It is encoded by 63 bp.

(9) Preparation of expression vector pUTE500K 'After digesting plasmid vector pBR322DNA (Takara Shuzo) with NdeI, blunt end year with DNA BLUNTING KIT (Takara Shuzo) and returning to circular with T4 DNA ligase (Takara Shuzo). The NdeI site was deleted with. This vector is then Eco
After digesting with RI and Nru I and blunting with the above DNA BLUNTING KIT, perform agarose electrophoresis according to a standard method,
Includes origin of replication with Quiaquick Gel Extraction Kit
A 3.4 kb DNA fragment was obtained. The obtained DNA is T4
After circularization with DNA ligase, Eco RI digestion was performed to linearize it.

Then, a promoter, an operator, etc. derived from the Escherichia coli lactose operon as shown below,
DNA containing ribosome binding site, expression control region such as terminator, Nde I cleavage site and sequencing primer (-21M13 FORWARD PRIMER) binding site
Sequence (The Operon, p277, Cold Spring Harbor Laborato
ry, 1980):

AATTCGGACCGGATCCGCTAGCTTTACATTATGCTTCCGGCTCGTATAA GCCTGGCCTAGGCGATCGAAATGTAATACGAAGGCCGAGCATATT

TGTCTGGAATTGTCAGCGGATAACAATTTCACACAGGAGGTTTCATATGC ACAGACCTTAACAGTCGCCTATTGTTAAAGTGTGTCCTCCAAAGTATACG

ATTGATCATTAATTAATAGCCCGCCTAATGAGCGGGCTTTTTTTTACTAG TAACTAGTAATTAATTATCGGGCGGATTACTCGCCCGAAAAAAAATGATC

TAGATCTCTGGCCGTCGTTTTACAGTCGACGGTACCG ATCTAGAGACCGGCAGCAAAATGTCAGCTGCCATGGCTTAA

Was synthesized using a DNA synthesizer Model 392 (manufactured by Applied Biosystems) and obtained as above.
The expression vector pUTE500 was constructed by ligating with the Eco RI fragment. An expression vector pUTE500K 'was prepared by ligating the kanamycin resistance gene (Pharmacia) digested at the Sal I site to the Sal I site of pUTE500.

(10) Preparation of Escherichia coli XL-1 Blue (pXylB500K ') Approximately 4 before and after the N-terminal of the xylulokinase gene was included.
An oligonucleotide (SEQ ID NO: 4) with 0 bases each and an oligonucleotide (SEQ ID NO: 5) with about 40 bases each before and after the C-terminal were purchased from DNA Agency and used as primers. These primers had an Nde I site incorporated into their sequences, and the products amplified by the PCR method were designed so that only the coding region could be obtained by digesting with Nde I. Using these primers, the plasmid DNA (pBK + 2-
Using the Sal I digest of 2) as a template, GeenAmp DNA PCR Reage
DNA containing a region encoding xylulokinase was amplified by PCR using ntKit (Takara Shuzo). After digesting this DNA with Nde I, the above expression plasmid pUTE500K '
It was inserted into the Nde I site of pXylB500K '. Escherichia coli XL-1 Blue (manufactured by Stratagene) was transformed with this plasmid according to a conventional method to obtain Escherichia coli XL-1 Blue (pXylB500K ′). This E. coli XL-1 Blue (pXylB500K ') was deposited on May 20, 1996 at the Institute of Biotechnology, Institute of Biotechnology, and the deposit number is FERM P-15636.

(11) Preparation of Escherichia coli XL-1 Blue (pXylE500K ') Similarly, for the xylose transporter, oligonucleotides of SEQ ID NO: 6 (N-terminal side) and SEQ ID NO: 7 (C-terminal side) were used (10). The xylose transporter was inserted into the Nde I site of the above expression plasmid pUTE500K 'in the same manner as in).
I got Escherichia coli XL-1 B
E. coli XL-1 transformed with lue (Stratagene)
I got Blue (pXylE500K '). This E. coli XL-1 Bl
ue (pXylE500K ′) was deposited with the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science, May 20, 1996, and the deposit number is FERM P-15637.

(12) Expression of enzyme activity etc. The above-mentioned Escherichia coli XL-1 Blue (pXylB500K ') was 1 m
50 ml TY medium containing M isopropyl-β-D-thiogalactopyranoside (1% bacto and lipton, 0.5% bacto yeast extract, 0.5% NaCl, pH
After shaking culture for 4 hours in 7), after centrifugation, 50m
5m after washing with M phosphate buffer (pH 7.5)
After suspending in 1 l of the same buffer, the bacterial cells were crushed at 10,000 psi with French pressure manufactured by Otake Seisakusho. The disrupted bacterial cell fluid was centrifuged at 30,000 xg for 10 minutes to obtain a crude enzyme solution. About this crude enzyme solution Appl. Environ. M
The activity was measured by the method described in icrobiol. 47 (10: 15-21, 1984) to obtain the activity of 0.5 μmol / min / mg protein.
E. coli having only pUTE500K 'as a control was E. coli.
The xylulokinase activity was 10% or less of that of XL-1 Blue (pXylB500K ').

On the other hand, Escherichia coli XL-1 Blue (pXylE500K ') was also cultivated in the same manner, the cells were washed with 50 mM phosphate buffer (pH 7.5), and then suspended in 0.5 ml of the same buffer. ． Bacteriol. 171: 17
93-1800 (1989), 14C-D
-The uptake capacity of xylose was measured. E. coli XL-1 Blue
(pXylE500K ') is 0.3μmol / min / mg dry cel
It showed the uptake activity of l. Escherichia coli having only pUTE500K 'as a control was E. coli XL-1 Blue (pXylE500K').
The xylose uptake activity was not more than%.

[0046]

Industrial Applicability According to the present invention, a gene for xylose metabolism enzyme can be provided. Then, by using this gene, it becomes possible to ferment xylose in any strain of Tetragenococcus halophilus, and it is extremely useful in pentose fermentation used for soy sauce brewing, grain fermentation, biomass and the like.

[0047]

[Sequence list]

SEQ ID NO: 1 Sequence length: 502 Sequence type: Amino acid Number of chains: Single strand Topology: Linear Sequence type: Protein (xylulokinase) Origin Biological name: Tetragenococcus halophilus (Tetrag
enococcus halophilus) strain name: I sequence: 1 5 10 15 Val Ile Glu Met Asp Tyr Val Leu Gly Leu Asp Leu Gly Thr Ser 20 25 30 Ser Leu Lys Gly Asp Leu Leu Asp Glu Ser Gly Lys Thr Val Cys 31 35 40 45 Val Ala Ser Thr Glu Tyr Glu Leu Leu Thr Pro Lys Ser Gly Tyr 50 55 60 Asn Glu Gln Asn Pro Asn Asp Trp Val Gln Ala Cys Tyr Arg Leu 61 65 70 75 Phe Glu Lys Met Ser Gln Lys Ile Ser Asp Phe Thr Ser Asn Leu 80 85 90 Ile Gly Ile Ser Phe Ser Gly Gln Met His Ser Leu Val Val Ala 91 95 100 105 Asp Glu Asp Gly Asn Ala Leu Arg Pro Ala Ile Leu Trp Asn Asp 110 115 120 Val Arg Thr Thr Lys Gln Cys Asn Asp Ile Thr Glu Asn Phe Gly 121 125 130 135 Glu Glu Ile Leu Lys Ile Thr Lys Asn Leu Val Leu Glu Gly Phe 140 145 150 Thr Leu Pro Lys Ile Leu Trp Ile Gln Glu Asn Glu Pro Glu Ile 151 155 160 165 Trp Lys Lys Val Arg Lys Ile Phe Leu Pro Lys Asp Tyr Leu Arg 170 175 180 Tyr Phe Leu Thr Gly Asn Phe Gln Met Asp Tyr Ser Asp Ala Ala 181 185 190 195 Gly Thr Leu Leu Met Asp Ile Lys Glu Lys Lys Trp Ser Gln Val 200 205 210 Ile L eu Lys Lys Tyr Asn Ile Pro Ile Glu Tyr Leu Pro Glu Leu 211 215 220 225 Val Asn Ser Phe Asp Phe Val Gly Asn Leu Asp Glu Asn Ile Lys 230 235 240 Lys Ser Tyr Gly Phe Lys Asn Asn Ile Lys Ile Phe Ala Gly Gly 241 245 250 255 Ala Asp Asn Ala Val Ser Ser Leu Gly Thr Gly Leu Asn Asp Tyr 260 265 270 Glu Thr Ala Ile Ala Ser Thr Gly Thr Ser Gly Val Phe Leu Ser 271 275 280 285 Leu Glu Asp Ser Ile His Asn Glu Tyr Asn Gly Lys Phe His Leu 290 295 300 Phe Asn His Ala Leu Pro Asp Lys Tyr Tyr Ser Met Gly Val Thr 301 305 310 315 Leu Ser Ala Gly Gln Ser Ile Ser Trp Phe Lys Asp Leu Ile Ala 320 325 330 Lys Ser Glu Ser Tyr Asp Lys Phe Leu Ser Asn Val Ser Asp Val 331 335 340 345 Pro Ile Gly Ser Asn Gly Leu Leu Phe Thr Pro Tyr Ile Ser Gly 350 355 360 Glu Arg Thr Pro Tyr Phe Asp Ser Gln Ile Arg Ala Ser Phe Ile 361 365 370 375 Gly Leu Asn Asp Thr His Ser Leu Asp Asp Leu Lys Arg Ala Val 380 385 390 Ile Glu Gly Val Thr Phe Ser Leu Lys Asp Ser Gln Val Leu Met 391 395 400 405 Glu Ser Leu Arg Glu Lys Glu Phe Arg Arg I le Ile Ser Ile Gly 410 415 420 Gly Gly Ala Lys Asn Lys Glu Trp Leu Gln Ile Gln Ala Asp Ile 421 425 430 435 Phe Asn Thr Gln Val Val Thr Leu Asp Thr Glu Glu Gly Pro Gly 440 445 450 Leu Gly Ala Ala Met Ile Ala Ala Met Gly Val Gly Trp Phe Glu 451 455 460 465 Thr Val Glu Glu Cys Val Ala His Thr Ile Thr Tyr Ile Asn Lys 470 475 480 Ile Asp Pro Ile Ser Glu Asn Val Ala Glu Tyr Lys Lys Ile Tyr 481 485 490 495 Glu Leu Tyr Thr Gln Val Tyr Pro Tyr Thr Lys Asp Ile Thr His 500 Ala Leu Ser Glu Asp Pro Val

SEQ ID NO: 2 Sequence length: 474 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Protein (xylose transporter) Origin Biological name: Tetragenococcus halophilus ( Tetrag
enococcus halophilus) Strain name: I Sequence: 1 5 10 15 Val Lys Ser His Pro Leu Thr Leu Thr Gln Asn Ile Met Asn Asn 20 25 30 Lys Gly Leu Asn Arg Tyr Val Ile Thr Ile Thr Leu Ile Ala Thr 31 35 40 45 Leu Gly Gly Leu Leu Phe Gly Tyr Asp Thr Ala Val Ile Ser Gly 50 55 60 Ala Glu Gln Ser Ile Gln Asn Asn Leu Val Asn Gly Val Gly Leu 61 65 70 75 Gly Ser Phe Ala His Gly Leu Thr Val Ser Ser Ala Leu Ile Gly 80 85 90 Cys Val Phe Gly Gly Leu Leu Ser Gly Ile Phe Ser Asn Asn Leu 91 95 100 105 Gly Arg Arg Asn Ser Leu Lys Leu Ala Gly Ser Leu Phe Leu Ile 110 115 120 Ser Ala Leu Gly Ser Ala Tyr Pro Glu Phe Leu Phe Phe Gln Ala 121 125 130 135 Gly Asp Ala Ser Val Gly Leu Leu Ile Val Phe Asn Leu Tyr Arg 140 145 150 Val Leu Gly Gly Ile Gly Val Gly Met Ala Ser Gly Ile Ser Pro 151 155 160 165 Thr Tyr Ile Gly Glu Ile Ala Pro Gly Lys Val Arg Gly Ser Leu 170 175 180 Val Ser Trp Tyr Gln Phe Ala Ile Ile Phe Gly Gln Leu Val Val 181 185 190 195 Tyr Phe Val Asn Trp Gly Ile Ala Thr Gly Gln Ser Pro Glu Trp 200 205 210 Val A sn Asp Ile Gly Trp Arg Phe Met Phe Ala Ser Glu Ala Ile 211 215 220 225 Pro Ala Ile Leu Phe Phe Ala Leu Leu Phe Tyr Val Pro Glu Thr 230 235 240 Pro Arg Tyr Leu Val Leu Lys Asn Asn Glu Glu Lys Ala Phe Asp 241 245 250 255 Val Leu Ser Lys Ile Asn Asn Ser Lys Glu Glu Ala Lys Asp Ile 260 265 270 Leu Thr Asp Ile Lys Gly Ser Leu Asn Thr Thr Glu Thr Ser Gly 271 275 280 285 Lys Leu Phe Ser Tyr Gly Lys Thr Val Val Ile Val Gly Val Leu 290 295 300 Leu Ser Ile Phe Gln Gln Phe Ile Gly Ile Asn Val Ala Leu Tyr 301 305 310 315 Tyr Ala Pro Arg Ile Phe Glu Ser Met Gly Ala Gly Gln Asn Ala 320 325 330 Ser Met Val Gln Thr Ile Ile Met Gly Ile Val Asn Val Thr Phe 331 335 340 345 Thr Tyr Val Ala Ile Arg Thr Val Asp Lys Trp Gly Arg Arg Pro 350 355 360 Leu Leu Leu Val Gly Ser Ile Gly Met Ala Ile Gly Met Phe Ala 361 365 370 375 Val Ala Leu Leu Ala Lys Asn Gly Val Leu Gly Ile Trp Thr Leu 380 385 390 Val Phe Ile Ile Val Tyr Thr Ala Ser Tyr Met Met Ser Trp Gly 391 395 400 405 Pro Ile Val Trp Val Leu Leu Ser Glu Ile P he Pro Asn Lys Ile 410 415 420 Arg Gly Gln Ala Met Ala Phe Ala Val Ala Ala Gln Trp Leu Ser 421 425 430 435 Asn Phe Phe Ile Ser Ser Thr Tyr Pro Ala Met Ile Asp Phe Ser 440 445 450 Gly Pro Leu Thr Tyr Gly Phe Tyr Gly Leu Met Cys Val Ile Ser 451 455 460 465 Ala Ile Phe Val Trp Lys Met Val Pro Glu Thr Lys Gly Lys Thr 470 Leu Glu Gln Leu Glu Asn Thr Trp Leu

SEQ ID NO: 3 Sequence length: 3430 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA Origin organism name: Tetragenococcus halophilus (Tetrag
enococcus halophilus) strain name: I SEQ: GGTATAGAAG GTATAGGTGA TTGAAATGGA TTATGTATTA GGTCTAGACT TAGGAACCAG 60 TTCGTTAAAA GGGGATCTGT TAGATGAAAG CGGTAAAACA GTTTGTGTAG CGTCAACAGA 120 ATATGAACTA CTAACGCCTA AAAGCGGTTA TAATGAGCAA AACCCAAACG ATTGGGTTCA 180 AGCATGTTAC CGTTTATTTG AAAAGATGTC CCAAAAGATT AGCGATTTTA CAAGCAACTT 240 AATAGGGATA AGCTTTTCAG GTCAGATGCA CTCCTTAGTT GTGGCCGACG AGGACGGCAA 300 TGCGTTAAGA CCTGCCATTC TTTGGAATGA TGTTCGTACA ACCAAACAAT GTAACGATAT 360 TACAGAAAAT TTTGGCGAAG AGATCTTAAA AATTACCAAA AATCTTGTTT TGGAAGGTTT 420 CACATTACCC AAAATATTAT GGATACAAGA AAATGAACCA GAGATTTGGA AGAAAGTAAG 480 GAAAATATTT TTACCCAAAG ACTATCTTAG GTATTTTTTA ACTGGAAATT TTCAGATGGA 540 TTATTCGGAT GCTGCGGGAA CTTTACTAAT GGATATCAAA GAAAAAAAGT GGTCACAGGT 600 TTTTGTTGGT AATTTAGATG AAAATATCAA AAAAAGTTAT GGATTTAAAA ATAATATCAA 720 AATATTTGCT GGTGGTGCTG ATAACGCTGT ATCTTCTTTA GGGACAGGAC TGAATGATTA 780 CGAGACTGCG ATAGCAAGCA CTGGTACTTC AGGAGTTTTT TTATCGTTAG AGGATAGTAT 840 TCATAATGAA TATAATGGAA AGTTCCATTT ATTTAATCAT GCTT TGCCAG ACAAGTATTA 900 TTCCATGGGT GTAACGTTAT CTGCAGGGCA AAGTATTTCT TGGTTTAAAG ACCTTATAGC 960 TAAGAGTGAG TCTTATGACA AGTTTTTAAG TAATGTGTCA GATGTTCCAA TTGGAAGTAA 1020 TGGTTTGTTA TTTACCCCTT ATATTTCGGG AGAAAGAACT CCATATTTTG ATAGCCAAAT 1080 AAGAGCTTCC TTCATTGGAT TAAACGATAC ACATTCATTG GATGATTTAA AAAGAGCAGT 1140 AATAGAAGGC GTAACATTTT CTTTAAAGGA CTCCCAAGTA TTGATGGAAT CCTTAAGAGA 1200 AAAGGAATTT AGACGAATAA TATCTATTGG CGGAGGAGCG AAAAATAAAG AATGGCTACA 1260 AATTCAAGCG GACATTTTTA ACACGCAAGT AGTGACTTTG GACACTGAAG AAGGGCCAGG 1320 ATTGGGAGCT GCCATGATTG CTGCCATGGG CGTTGGCTGG TTTGAAACAG TTGAAGAATG 1380 TGTGGCACAT ACTATAACTT ATATCAATAA AATTGACCCC ATTTCAGAAA ATGTCGCAGA 1440 GTATAAAAAA ATTTATGAAT TGTATACTCA AGTTTATCCA TATACAAAAG ATATCACTCA 1500 TGCATTAAGT GAAGACCCGG TTTAAATAAG GAATTCAAGA TATTAGGTAG CTACTACTCA 1560 ATAAACTAAT CATCGCTACT GTAAGCACTC AAGCGAAATT AAGTGGATTA CTATTTTTGT 1620 TGTACATTGA TAGTGAGCGA TACAACCACG AGGGTGGTTT CATTAACATA GACAGCTCTT 1680 ATACAAGAAA ATATGTTGTC TTGGCAGTTT TGTGGAAGTA GCTACCTTTT C CTTGATTGA 1740 TTAAATAAAG CATTGGAACT TTTTAAAACA ACTGTTAATG AGGTACGGAT AGTTATCAAG 1800 GGCTTATTAT CAAATTTACA AGAGGGAGGG ATAGAAGAGA AGTGAAGTCA CACCCGCTTA 1860 CGCTTACTCA AAATATTATG AATAATAAAG GATTAAATCG CTATGTTATT ACAATTACCT 1920 TAATTGCAAC TTTGGGAGGT CTATTATTCG GTTATGATAC TGCTGTTATA TCTGGTGCTG 1980 AACAATCTAT CCAGAATAAT CTAGTTAATG GAGTAGGTTT AGGTTCCTTT GCTCACGGGT 2040 TAACAGTTTC TAGCGCACTT ATTGGTTGTG TTTTTGGAGG TCTATTATCT GGAATATTTT 2100 CAAATAATTT GGGGAGAAGA AACTCTTTAA AATTAGCAGG TAGCCTCTTT TTAATTTCCG 2160 CATTGGGGTC TGCGTACCCA GAATTTTTGT TCTTTCAAGC CGGTGATGCA TCAGTTGGTC 2220 TTCTAATTGT ATTTAATTTA TACCGCGTTC TTGGTGGTAT CGGTGTTGGA ATGGCTTCCG 2280 GTATATCTCC CACATATATT GGTGAAATTG CTCCTGGAAA AGTTCGTGGC AGCCTAGTTT 2340 CGTGGTATCA GTTTGCTATT ATTTTTGGAC AATTAGTTGT ATATTTCGTA AATTGGGGAA 2400 TTGCCACCGG ACAATCACCA GAATGGGTGA ATGATATTGG ATGGAGATTT ATGTTTGCTT 2460 CAGAAGCAAT ACCGGCTATT TTATTTTTTG CTTTGTTGTT TTATGTACCT GAAACACCTA 2520 GATACTTAGT GTTAAAAAAT AACGAAGAAA AAGCTTTTGA CGTTTTATCT AAAATTA ACA 2580 ACTCTAAAGA AGAGGCAAAA GATATATTAA CTGATATTAA AGGCTCTCTA AATACAACAG 2640 AAACGAGTGG TAAGTTATTT TCTTATGGTA AAACTGTAGT CATTGTAGGT GTTTTACTCT 2700 CAATATTTCA GCAATTCATA GGTATTAACG TTGCTTTATA TTATGCACCA AGAATATTTG 2760 AAAGTATGGG CGCCGGCCAA AATGCCTCGA TGGTACAGAC AATCATTATG GGAATTGTTA 2820 ACGTGACATT TACCTATGTT GCGATAAGAA CTGTAGATAA ATGGGGGAGA AGACCGTTGT 2880 TGTTAGTTGG ATCAATAGGG ATGGCTATTG GTATGTTCGC TGTTGCGTTG CTTGCGAAAA 2940 ATGGTGTGTT GGGTATTTGG ACGTTAGTCT TTATCATAGT TTATACGGCC TCCTATATGA 3000 TGTCGTGGGG ACCGATTGTT TGGGTGTTAC TTTCTGAAAT ATTCCCTAAC AAAATCAGAG 3060 GACAAGCAAT GGCATTTGCA GTTGCTGCTC AATGGCTATC AAACTTCTTT ATTTCATCAA 3120 CCTATCCTGC AATGATAGAC TTTAGTGGAC CGCTAACTTA TGGATTTTAT GGTTTGATGT 3180 GTGTTATTTC CGCAATTTTT GTATGGAAGA TGGTCCCAGA AACAAAAGGA AAGACGCTTG 3240 AACAATTAGA AAATACTTGG TTATAAGAAA AAATGGAAAA AAATAAAAAT AGTAATAAAC 3300 CTCCTGAATG ATTTATAAAT TTTTCTAAGG TTTATTCAAA CCGTGGCATA CTCTTTTTTC 3360 TAAGGGGGTT CATTGCTTCA CCTTCTGGGT GTACAAAAAA GAACCGACTT CGTGTTATTG 34 20 TAAAGTCGAC .......... .......... .......... .......... ........ .. 3430

SEQ ID NO: 4 Sequence length: 44 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Sequence characteristics: Synthetic DNA primer Sequence: 5'GGTATAGAAGGTACATATGATTGAAATGGATTATGTATTAGGTC3 '

SEQ ID NO: 5 Sequence length: 51 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Sequence characteristics: Synthetic DNA primer Sequence: 5'GAGTAGTAGCTACCTCATATGTTGAATTCCTTATTTAAACCGGGTCTTCAC3 '

SEQ ID NO: 6 Sequence length: 51 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Sequence characteristics: Synthetic DNA primer Sequence: 5'CAAGAGGGAGGGATAGAAGACATATGAAGTCACACCCGCTTACGCTTACTC3 '

SEQ ID NO: 7 Sequence length: 52 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Sequence characteristics: Synthetic DNA primer Sequence: 5'TTATTTTGTTCCATATGTTTCTTATAACCAAG
TATTTTCTAATTTGTTCAAGCG3 '

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location (C12N 9/12 C12R 1:19)

Claims (2)

[Claims] 1. An amino acid that provides a xylose-metabolizing enzyme activity comprising a xylulokinase having substantially the amino acid sequence shown in SEQ ID NO: 1 and / or a xylose transporter having substantially the amino acid sequence shown in SEQ ID NO: 2. A gene that encodes a sequence. 2. The gene according to claim 1, wherein the gene encoding an amino acid sequence that brings about the xylose metabolism enzyme activity is the DNA sequence shown in SEQ ID NO: 3.