1. Field of the Invention
The present invention relates to phosphohexuloisomerase and a DNA coding for it. More precisely, the present invention related to phosphohexuloisomerase derived from a thermotolerant bacterium, Bacillus brevis, and a DNA coding for it.
2. Description of the Related Art
Among organisms that can utilize single carbon (C1) compounds such as methane and methanol as a carbon source (methylotrophs), there are known those having the ribulose monophosphate (RuMP) pathway as a pathway for metabolizing such compounds. Important key enzymes of this pathway are hexulose phosphate synthase (HPS, 3-hexulose-6-phosphate synthase), which catalyzes the initial reaction of the ribulose monophosphate pathway, and phosphohexuloisomerase (PHI, phospho-3-hexuloisomerase), which catalyzes the subsequent reaction.
By the way, biochemical substances in which specific position of a target compound molecule is labeled with a stable isotope, carbon 13 (13C), are useful for study of biological metabolic pathway. Furthermore, it has recently become a very important technique to investigate behaviors of metabolic products in living bodies by using carbon 13-NMR techniques in diagnosis of various diseases and daily health examination. For such novel techniques, it is necessary and desired to provide compounds labeled at a certain target position with carbon 13 at a low cost.
As one of systems for producing such target compounds as mentioned above, a method can be conceived, in which a series of enzymes are prepared for synthesizing labeled D-fructose 6-phosphate using labeled formaldehyde and ribulose 5-phosphate, and a target labeled compound is efficiently prepared in a reaction system utilizing the enzymes. Hexulose phosphate synthase, which is an enzyme initially acts in the reaction system, has been isolated from several kinds of microorganisms, and some of its characteristics have been elucidated. Such microorganisms include, for example, Methylomonas capsulatus (J. R. Quayle, Methods in Enzymology, 188, p.314, 1990), Methylomonas M15 strain (Methods in Enzymology, 188, p.319, 1990), Methylomonas aminofaciens 77a strain (Biochim. Biophys. Acta., 523, p.236, 1978), Mycobacterium gastri MB19 (Methods in Enzymology, 188, p.393, 1990), and Acetobacter methanolicus MB58 (Methods in Enzymology, 188, p.401, 1990).
Further, as for phosphohexuloisomerase, it has been partially purified from Methylomonas aminofaciens 77a strain (Agric. Biol. Chem., 41 (7), p1133, 1977), and a purified enzyme and a gene coding for it were isolated from a gram-positive facultative methanol assimilating bacterium, Mycobacterium gastri (Japanese Patent Laid-open Publication (Kokai) No. 11-127869).
Enzymes and proteins produced by thermotolerant bacteria are generally stable at a high temperature, and most of them are also stable against pH variation and organic solvents. Therefore, applications thereof have been highly developed as diagnostic agents, industrial catalysts and so forth. As a C1 metabolic system enzyme of thermotolerant methanol assimilating bacteria, only hexulose phosphate synthase has been purified from Bacillus methanolicus C1 strain (Methods in Enzymology, 188, p.393, 1990), and its detailed structure and gene therefor are unknown. On the other hand, as for phosphohexuloisomerase of thermotolerant bacteria, not only the structure of enzyme protein and gene therefor, but also purification of the enzyme have not been reported.
The inventors of the present invention found that, in the course of cloning of a gene coding for hexulose phosphate synthase (henceforth also referred to as xe2x80x9chpsxe2x80x9d) of Bacillus brevis S1 strain, a gene coding for PHI (henceforth also referred to as xe2x80x9cphixe2x80x9d) existed in the DNA fragment containing hps. And they isolated the phi gene, introduced this gene into an Escherichia coli cell, and examined activity of the expression product to confirm that the gene coded for PHI. Thus, they accomplished the present invention.
That is, the present invention provides the followings.
(1) A DNA coding for a protein defined in the following (A) or (B):
(A) a protein having the amino acid sequence of SEQ ID NO: 3 shown in Sequence Listing,
(B) a protein having the amino acid sequence of SEQ ID NO: 3 shown in Sequence Listing including substitution, deletion, insertion or addition of one or several amino acid residues and having phosphohexulose isomerase activity.
(2) The DNA according to (1), which is a DNA defined in the following (a) or (b):
(a) a DNA containing a nucleotide sequence consisting of at least the residues of nucleotide numbers 1149-1700 of the nucleotide sequence of SEQ ID NO: 1 shown in Sequence Listing,
(b) a DNA which is hybridizable with a nucleotide sequence consisting of at least the residues of nucleotide numbers 1149-1700 of the nucleotide sequence of SEQ ID NO: 12 shown in Sequence Listing under a stringent condition, and codes for a protein having phosphohexulose isomerase activity.
(3) A cell into which a DNA according to (1) or (2) is introduced in such a manner that phosphohexulose isomerase encoded by the DNA can be expressed.
(4) A method for producing phosphohexulose isomerase, comprising culturing the cell according to (3) in a medium to produce and accumulate phosphohexulose isomerase in culture, and collecting the phosphohexulose isomerase from the culture.
(5) A protein defined in the following (A) or (B):
(A) a protein having the amino acid sequence of SEQ ID NO: 3 shown in Sequence Listing,
(B) a protein having the amino acid sequence of SEQ ID NO: 3 shown in Sequence Listing including substitution, deletion, insertion or addition of one or several amino acid residues and having phosphohexulose isomerase activity.
According to the present invention, a DNA coding for phosphohexuloisomerase is obtained, and this enables efficient production of that enzyme. As a result, it becomes possible to provide labeled substances that are important and required for medicine or biochemical basic research in large quantities at low cost.
Hereafter, the present invention will be explained in detail.
 less than 1 greater than  DNA of the Present Invention
The DNA of the present invention was found in the DNA fragment containing hps gene of Bacillus brevis S1 strain adjacent to and downstream from the hps gene, and it can be isolated and obtained from chromosomal DNA of Bacillus brevis. Specifically, as shown in the examples described later, the DNA of the present invention was obtained from chromosomal DNA of Bacillus brevis as follows.
First, HPS is purified from Bacillus brevis. As the Bacillus brevis, the Bacillus brevis S1 strain can be mentioned. This strain is subcultured at NCIMB (The National Collections of Industrial and Marine Bacteria) with the accession number of NCIMB12524.
HPS can be purified from cell free extract of the S1 strain by Q-Sepharose column chromatography, Buthyl-Toyopearl column chromatography and Superdex 200 column chromatography to such a degree that it can be detected as a single band in SDS-PAGE. In each purification step, HPS activity can be measured by the method described in Methods in Enzymology, vol. 188, 397-401 (1990).
A partial amino acid sequence of the purified HPS is determined, and oligonucleotide primers for PCR (polymerase chain reaction) are synthesized based on the obtained amino acid sequence information. Then, PCR is performed by using genomic DNA prepared from the Bacillus brevis S1 strain as a template. The genomic DNA can be obtained by the method of Saito et al. (described in Biochim. Biophys. Acta, 72, 619-629 (1963)). If the oligonucleotides having the nucleotide sequences of SEQ ID NOS: 7 and 8 shown in Sequence Listing are used as primers, a DNA fragment of about 400 bp will be obtained by the above PCR.
Then, based on the nucleotide sequence of the hps fragment obtained as described above, a DNA fragment containing the hps gene in its full length is obtained from Bacillus brevis S1 strain chromosomal DNA by, for example, the inverted PCR method (Genetics, vol. 120, pp.621-623, 1988) using oligonucleotides having the nucleotide sequences of SEQ ID NOS: 9 and 10 as primers.
At first, the inventors of the present invention attempted to screen a genomic library of Bacillus brevis S1 strain by using the aforementioned hps fragment of about 400 bps as a probe. However, although the possible cause was unknown, probably because a problem resided in the ligation of the chromosomal DNA fragment to the vector, colonies having a number of genomic libraries sufficient for the screening could not be formed, and thus they had to give up use of the ordinary method.
Therefore, they attempted the cloning by the inverted PCR technique as described above, and successfully obtained a DNA fragment containing the hps gene. The result of nucleotide sequence determination for about 1.8 kb in the clone fragment in a length of about 3 kb obtained as described above is shown in Sequence Listing as SEQ ID NO: 1. In this region, two open reading frames (orfs) were contained. The amino acid sequences encoded by each orf are shown in SEQ ID NOS: 2 and 3 from the 5xe2x80x2 end side. Since the first orf among these completely coincided to a partial amino acid sequence of HPS, it was demonstrated to be hps. On the other hand, the second orf was confirmed to be phi, i.e., the DNA of the present invention, by investigating the activity of a protein obtained by expressing this orf.
When homology searching was performed for the nucleotide sequence of phi and the amino acid sequence encoded thereby by using commercially available software (GENETYX), they showed 65.6% of homology on the nucleotide level and 64.3% of homology on the amino acid level to ykcF of Bacillus subtillis. The homology was calculated as a ratio of the number of exactly the same amino acid residues in yckF and phi to the total number of amino acid residues encoded by yckF.
As described above, while the DNA of the present invention was discovered by chance in connection with the purification of HPS and isolation of hps, the DNA of the present invention was obtained by expressing the second orf and confirming the activity of the expressed product based on a conception that the second orf should code for phi.
The DNA of the present invention was obtained as described above. However, since its nucleotide sequence and the amino acid sequence encoded thereby were elucidated by the present invention, the DNA of the present invention can then be obtained from a genomic DNA library of a thermotolerant bacterium belonging to the genus Bacillus, for example, Bacillus brevis S1 strain, by hybridization utilizing an oligonucleotide produced based on the nucleotide sequence or amino acid sequence as a probe. The DNA of the present invention can also be obtained by performing PCR utilizing the aforementioned oligonucleotide as a primer and genomic DNA of a thermotolerant bacterium belonging to the genus Bacillus as a template.
Methods for construction of genomic DNA library, hybridization, PCR, preparation of plasmid DNA, digestion and ligation of DNA, transformation and so forth are described in by Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1.21 (1989).
The Escherichia coli JM109/pKPS1 harboring a plasmid pKPS1 containing the DNA of the present invention and expressing PHI under control of tac promoter, which was obtained in the examples mentioned later and given a private number of AJ13707, was deposited on Jul. 5, 2000 at the National Institute of Bioscience and Human Technology of Agency of Industrial Science and Technology (currently, the independent administrative corporation, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary)(Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, postal code: 305-5466) as the accession number of FERM P-17952, and transferred from the original deposit to international deposit based on Budapest Treaty on Jun. 25, 2001, and has been deposited as the accession number of FERM BP-7639.
The DNA of the present invention may code for PHI including substitution, deletion, insertion or addition of one or several amino acid residues at one or a plurality of positions, provided that the activity of encoded PHI is not deteriorated. The number of xe2x80x9cseveralxe2x80x9d amino acid residues differs depending on positions or types of amino acid residues in the three-dimensional structure of the protein. However, the encoded PHI may be one showing homology of 65% or more, preferably 80% or more, to the total amino acid sequence constituting PHI and having the PHI activity. Specifically, the number of xe2x80x9cseveralxe2x80x9d amino acid residues is preferably 2-60, more preferably 2-30, further preferably 2-10.
A DNA coding for substantially the same protein as PHI described above can be obtained by, for example, modifying the nucleotide sequence by, for example, the site-directed mutagenesis method so that the amino acid sequence should involve substitution, deletion, insertion or addition of one or more amino acid residues at a specified site. Such a DNA modified as described above may also be obtained by a conventionally known mutation treatment. The mutation treatment includes a method of treating DNA coding for PHI in vitro, for example, with hydroxylamine, and a method for treating a microorganism, for example, a bacterium belonging to the genus Escherichia, harboring a DNA coding for PHI with ultraviolet irradiation or a mutating agent usually used for mutation treatment such as N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine (NTG) and nitrous acid.
The substitution, deletion, insertion, addition or inversion of nucleotide as described above also includes a naturally occurring mutant or variant on the basis of, for example, individual difference or difference in species or genus of microorganisms that harbor phi.
A DNA coding for substantially the same protein as PHI described above can be obtained by expressing such a DNA having a mutation as described above in a suitable cell, and examining the PHI activity of the expression product. A DNA coding for substantially the same protein as PHI can also be obtained by isolating a DNA hybridizable with a DNA having, for example, the nucleotide sequence corresponding to nucleotide numbers of 1149-1700 of the nucleotide sequence shown in SEQ ID NO: 1 or a probe that can be prepared from the nucleotide sequence under a stringent condition, and coding for a protein having the PHI activity from a DNA coding for PHI including a mutation or a cell harboring it. The xe2x80x9cstringent conditionxe2x80x9d referred to herein is a condition under which so-called specific hybrid is formed, and non-specific hybrid is not formed. It is difficult to clearly express this condition by using any numerical value. However, for example, the stringent condition includes a condition under which DNA""s having high homology, for example, DNA""s having homology of not less than 70% are hybridized with each other, and DNA""s having homology lower than the above level are not hybridized with each other. Alternatively, the stringent condition is exemplified by a condition under which DNA""s are hybridized with each other at a salt concentration corresponding to an ordinary condition of washing in Southern hybridization, i.e., 1xc3x97SSC, 0.1% SDS, preferably 0.1xc3x97SSC, 0.1% SDS, at 60xc2x0 C.
As the probe, a partial sequence of the phi gene can also be used. Such a probe can be produced by PCR (polymerase chain reaction) using oligonucleotides produced based on the nucleotide sequence of each gene as primers and a DNA fragment containing each gene as a template. When a DNA fragment in a length of about 300 bp is used as the probe, the washing condition for the hybridization may consists of 50xc2x0 C., 2xc3x97SSC and 0.1% SDS.
Genes hybridizable under such a condition as described above include those having a stop codon generated in a coding region of the genes, and those having no activity due to mutation of active center. However, such mutants can be readily removed by ligating each of the genes with a commercially available activity expression vector, and measuring the PHI activity by the method described above.
 less than 2 greater than  Production of Hexulose Phosphate Isomerase
PHI can be produced by allowing expression of the aforementioned DNA of the present invention using a suitable host-vector system.
As the host for the expression of the phi gene, there can be mentioned various prokaryote cells including Escherichia coli and various eucaryote cells including Saccharomyces cerevisiae, animal cells and plant cells. Among these, prokaryote cells, especially Escherichia coil cells, are preferred.
As the vector for introducing the phi gene into the aforementioned host, there can be mentioned, for example, pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, pMW218 and so forth. Other than these, a vector of phage DNA can also be used. The phi gene can be introduced into the host by transforming the host with a recombinant vector obtained by ligating the phi gene to any one of those vectors. The phi gene may also be introduced into genome of the host by a method using transduction, transposon (Berg, D. E. and Berg C. M., Bio/Technol., 1, 417 (1983)), Mu phage (Japanese Patent Laid-open Publication No. 2-109985/1990) or homologous recombination (Experiments in Molecular Genetics, Cold Spring Harbor Lab. (1972)).
Further, in order to obtain efficient expression of the phi gene, a promoter functioning in the host cell such as lac, trp and PL may be ligated to the DNA sequence coding for PHI in its upstream region. If a vector containing a promoter is used as the vector, the ligation of the phi gene, vector and promoter can be performed at once. As such a vector, pKK 223-3 containing tac promoter (Pharmacia) can be mentioned.
For the transformation, there can be used, for example, a method of treating recipient cells with calcium chloride so as to increase the permeability for DNA, which has been reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)); and a method of preparing competent cells from cells which are at the growth phase followed by introducing the DNA thereinto, which has been reported for Bacillus subtilis (Duncan, C. H., Wilson, G. A. and Young, F. E., Gene, 1, 153 (1977)). In addition to these, also employable is a method of making DNA-recipient cells into protoplasts or spheroplasts, which can easily take up recombinant DNA, followed by introducing the recombinant DNA into the cells, which method is known to be applicable to Bacillus subtilis, actinomycetes and yeasts (Chang, S. and Choen, S. N., Molec. Gen. Genet., 168, 111 (1979); Bibb, M. J., Ward, J. M. and Hopwood, O. A., Nature, 274, 398 (1978); Hinnen, A., Hicks, J. B. and Fink, G. R., Proc. Natl. Sci. USA, 75, 1929 (1978)). The method for transformation may be suitably selected from these methods depending on the cells used as the host.
Although the phi gene may be any one so long as it shows the PHI activity when it is expressed, it is preferably a gene containing a DNA coding for the amino acid sequence of SEQ ID NO: 3 shown in Sequence Listing, or a DNA containing the nucleotide residues of the nucleotide numbers 1149-1700 in the nucleotide sequences of SEQ ID NO: 1 shown in of Sequence Listing. Further, as mentioned above, it may be one containing a DNA coding for PHI including substitution, deletion, insertion, addition or inversion of one or several amino acid residues at one or a plurality of positions, so long as the activity of the encoded PHI is not deteriorated.
PHI can be produced by culturing a cell introduced with the phi gene as described above in a medium to produce and accumulated PHI in culture, and collecting PHI from the culture. The medium used for the culture can be suitably selected depending on a host to be used. When Escherichia coli is used as the host and phi is expressed with the aid of tac promoter, if the host is cultured in a medium such as LB medium at 37xc2x0 C., IPTG (isopropyl-xc3xa2-D-thiogalactopyranoside), which is an inducer for the tac promoter, is added at a final concentration of 0.5 mM several hours after the start of the culture, and the culture is further continued, PHI is accumulated in the cells. When extracellular secretion of PHI is allowed by using a suitable secretion system, PHI is accumulated in the medium.
PHI produced as described above can be purified from cell extract or medium by using usual purification methods for enzymes such as ion exchange chromatography, gel filtration chromatography, adsorption chromatography and solvent precipitation as required.
PHI obtained by the present invention can be used for producing [1-13C] D-glucose 6-phosphate from methanol labeled with carbon 13. The preparation of this [1-13C] D-glucose 6-phosphate can be performed as follows, for example. Methanol is oxidized into formaldehyde by using alcohol oxidase prepared from methanol assimilating yeast, Candida boidinii. The obtained formaldehyde is condensed with ribulose 5-phosphate through aldol condensation by the action of HPS to form arabino-3-hexulose 6-phosphate. In this case, since ribulose 5-phosphate is unstable, ribose 5-phosphate is isomerized into ribulose 5-phosphate by the action of phosphoriboisomerase in the same reaction system for use in the HPS reaction. The arabino-3-hexulose 6-phosphate produced in the aforementioned reaction is converted into fructose 6-phosphate by the action of PHI, which is further converted into glucose 6-phosphate by the action of glucose 6-phosphate isomerase. Because the PHI content is markedly lower than the HPS content in general, it is difficult to utilize PHI for the aforementioned reaction in most of cases. Further, it is considered that the reaction can be continued for a long period of time by using PHI of a thermotolerant bacterium. Since phi of a thermotolerant bacterium was isolated and a method for efficiently producing PHI was provided by the present invention, it became possible to stably perform the aforementioned reaction for practical use.
Hereafter, the present invention will be explained more specifically with reference to the following examples.
First, the method for measurement of HPS activity (Methods in Enzymology, vol. 188, 397-401 (1990)) used in the examples will be explained.
[Method for Measurement of HPS Activity]
To 0.15 ml of water, 0.05 ml each of the following Solutions A to E were added and mixed in a cuvette (d=1.0 cm), and preliminarily heated at 30xc2x0 C. for about 3 minutes. To the cuvette, 0.1 ml of 10 mM formaldehyde solution was added to start the reaction. The reaction was allowed at 30xc2x0 C. for 5 minutes, and then 0.1 ml of 0.5 N hydrochloric acid was added to the mixture to stop the reaction. The reaction mixture was diluted 20 times and added with 2 ml of Nash reagent (described in Biochem. J., 55, 416, 1953), and decrease of formaldehyde in the reaction mixture was measured. In a control experiment, water was used instead of the ribose 5-phosphate solution.
[Reagents]
A: 500 mM Potassium phosphate buffer, pH 7.5
B: 50 mM Magnesium chloride aqueous solution
C: 50 mM Ribose 5-phosphate aqueous solution
D: 100 U/ml of phosphoriboisomerase solution
E: Enzyme preparation (50 mM phosphate buffer (pH 7.5) containing 1 mM DTT)