# Patent

## Title
Purine-derived substance-producing bacterium and a method for producing a purine-derived substance

## Abstract

A purine-derived substance is produced by culturing a bacterium belonging to the genus Bacillus which has an ability to produce a purine-derived substance and has been modified so that enzymatic activity of transaldolase is decreased in a medium to cause accumulation of a purine-derived substance in the medium or cells, and collecting the purine-derived substance from the medium or cells.

## Background
This application is a continuation under 35 U.S.C. §120 to PCT Patent Application No. PCT/JP2007/058357, filed on Apr. 17, 2007, which claims priority under 35 U.S.C. §119 to Japanese Patent Application No. 2006-119324, filed Apr. 24, 2006, both of which are incorporated by reference. The Sequence Listing in electronic format filed herewith is also hereby incorporated by reference in its entirety (File Name: US-377_Seq_List; File Size: 69 KB; Date Created: Oct. 21, 2008).


BACKGROUND OF THE INVENTION
1. Technical Field
The present invention relates to a method for producing purine-derived substances such as purine nucleotides purine nucleosides. Purine nucleotides typically include 5′-inosinic acid and 5′-guanylic acid, purine nucleosides typically include inosine and guanosine. Purine nucleosides are important for their use as starting materials for the synthesis of purine nucleotides, and so forth. Bacillus bacteria can be used in the methods described herein. Purine-derived substances are useful as seasonings, drugs, raw materials thereof, and so forth.
2. Background Art
Methods for producing inosine and guanosine by fermentation using adenine auxotrophic strains of Bacillus bacteria have been reported. Derivatives of these bacteria which are made resistant to various drugs such as purine analogues have also been reported (Japanese Patent Publication (KOKOKU) No. 38-23099, Japanese Patent Publication No. 54-17033, Japanese Patent Publication No. 55-2956, Japanese Patent Publication No. 55-45199, Japanese Patent Publication No. 57-14160, Japanese Patent Publication No. 57-41915, Japanese Patent Laid-open (KOKAI) No. 59-42895, and Japanese Patent Laid-open No. 2004-242610). Microorganisms of the genus Brevibacterium have also been reported to be useful for production of inosine and guanosine by fermentation (Japanese Patent Publication No. 51-5075, Japanese Patent Publication No. 58-17592, and Agric. Biol. Chem., 1978, 42, 399-405).
Such mutant strains are typically obtained by treating the microorganism with ultraviolet irradiation or nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine), and selecting the mutant with the desired properties using a suitable selection medium.
Furthermore, strains which produce purine-derived substances have also been bred using genetic engineering techniques in Bacillus bacteria (Japanese Patent Laid-open No. 58-158197, Japanese Patent Laid-open No. 58-175493, Japanese Patent Laid-open No. 59-28470, Japanese Patent Laid-open No. 60-156388, Japanese Patent Laid-open No. 1-27477, Japanese Patent Laid-open No. 1-174385, Japanese Patent Laid-open No. 3-58787, Japanese Patent Laid-open No. 3-164185, Japanese Patent Laid-open No. 5-84067, and Japanese Patent Laid-open No. 5-192164), Brevibacterium bacteria (Japanese Patent Laid-open No. 63-248394), and Escherichia bacteria (International Patent Publication WO99/03988). Specifically, for example, a method for efficiently producing nucleic acid-derived compounds such as hypoxanthine, uracil, guanine and adenine with a Bacillus bacterium in which the gene (purR) encoding a purine operon repressor is disrupted has been disclosed (U.S. Pat. No. 6,284,495).
In Bacillus subtilis, the purine operon repressor as described above is known to regulate the genes of the purine operon. The purine operon repressor also regulates the purA gene, which is involved in AMP biosynthesis (Proc. Natl. Acad. Sci. USA, 1995, 92, 7455-7459), the glyA gene, which is involved in formyltetrahydrofolic acid biosynthesis (J. Bacteriol., 2001, 183, 6175-6183), the pbuG gene, which encodes the transporter of hypoxanthine/guanine (J. Bacteriol., 2003, 185, 5200-5209), and so forth.
Furthermore, a microorganism which is made auxotrophic for adenine by disruption of the succinyl-AMP synthase gene (purA) and purR genes, and suppression of the decomposition of inosine into hypoxanthine by disruption of the purine nucleoside phosphorylase gene (deoD), has also been reported, as well as a method for producing inosine using this microorganism (see Japanese Patent Laid-open No. 2004-242610).
Transaldolase is one of the enzymes of the pentose phosphate pathway, which catalyzes the reversible reaction which generating D-erythrose-4-phosphate and D-fructose-6-phosphate from sedoheptulose-7-phosphate and D-glyceraldehyde-3-phosphate. There is not much known about the relationship between this enzyme and the biosynthetic pathway of purine-derived substances, and there have been no reports of an attempt to breed bacteria able to produce purine-derived substances by reducing the activity of this enzyme.
SUMMARY OF THE INVENTION
The present invention describes a Bacillus bacterium suitable for fermentative production of purine-derived substances such as purine nucleosides and/or purine nucleotides, and provides a method for producing a purine-derived substance using such a bacterium.
It was found that when the enzymatic activity of transaldolase of the pentose phosphate pathway is decreased in a Bacillus bacterium, the ability of the bacterium to produce purine nucleosides or purine nucleotides is improved.
The present invention thus provides the following:
It is an aspect of the present invention to provide a bacterium belonging to the genus Bacillus which is able to produce a purine-derived substance, wherein the bacterim has been modified to decrease the enzymatic activity of transaldolase.
It is a further aspect of the present invention to provide the bacterium as described above, wherein the purine-derived substance is purine nucleoside selected from the group consisting of inosine, xanthosine, guanosine, and adenosine.
It is a further aspect of the present invention to provide the bacterium as described above, wherein the purine-derived substance is purine nucleotide selected from the group consisting of inosinic acid, xanthylic acid, guanylic acid, and adenylic acid.
It is a further aspect of the present invention to provide the bacterium as described above, wherein the transaldolase activity is decreased by disrupting the gene encoding transaldolase, or reducing the expression of the gene encoding transaldolase.
It is a further aspect of the present invention to provide the bacterium as described above, wherein said gene encodes transaldolase selected from the group consisting of:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 1,
(B) a protein comprising an amino acid sequence of SEQ ID NO: 1, but which includes substitutions, deletions, insertions, additions, or inversions of one or several amino acid residues and has transaldolase activity, and
(C) combinations thereof.
It is a further aspect of the present invention to provide the bacterium as described above, which is further modified to increase phosphoribosyl pyrophosphate synthetase activity.
It is a further aspect of the present invention to provide the bacterium as described above, which has been further modified to increase the expression of the purine operon.
It is a further aspect of the present invention to provide the bacterium as described above, wherein said expression is increased by disrupting the purR gene or deleting a part of the attenuator region of the purine operon, wherein said purR gene encodes a repressor of the purine operon.
It is a further aspect of the present invention to provide the bacterium as described above, which has been further modified to decrease purine nucleoside phosphorylase activity.
It is a further aspect of the present invention to provide the bacterium as described above, which has been further modified to decrease fructose bisphosphatase activity.
It is a further aspect of the present invention to provide the bacterium as described above, which has been further modified to decrease IMP dehydrogenase activity.
It is a further aspect of the present invention to provide the bacterium as described above, which is Bacillus subtilis. 
It is another aspect of the present invention to provide a method for producing a purine-derived substance, which comprises culturing the Bacillus bacterium as described above in a medium, and collecting the purine-derived substance from the bacterium or medium.
It is a further aspect of the present invention to provide the method as described above, wherein the purine-derived substance is a purine nucleoside or a purine nucleotide.
It is a further aspect of the present invention to provide the method as described above, wherein the purine-derived substance is a purine nucleoside selected from the group consisting of inosine, xanthosine, guanosine, and adenosine.
It is a further aspect of the present invention to provide the method as described above, wherein the purine-derived substance is a purine nucleotide selected from the group consisting of inosinic acid, xanthylic acid, guanylic acid, and adenylic acid.
It is a further aspect of the present invention to provide a method for producing a purine nucleotide, which comprises
(A) producing a purine nucleoside by the method as described above,
(B) reacting the purine nucleoside with a phosphate donor selected from the group consisting of polyphosphoric acid, phenyl phosphate, and carbamyl phosphate, and a microorganism which is able to produce a nucleoside-5′-phosphoric acid ester or acid phosphatase to produce a purine nucleotide, and
(C) collecting the purine nucleotide.


DESCRIPTION OF THE PREFERRED EMBODIMENTS
<1> Bacillus Bacterium
(I) Imparting the Ability to Produce a Purine-Derived Substance
The Bacillus bacterium is able to produce a purine-derived substance and has been modified to decrease the enzymatic activity of transaldolase.
The term “purine-derived substance” means a substance having a purine skeleton, and examples include purine nucleosides, purine nucleotides, and so forth. The purine nucleosides include inosine, xanthosine, guanosine, adenosine, and so forth, and the purine nucleotides include 5′-phosphoric acid esters of purine nucleosides, for example, inosinic acid (inosine-5′-phosphate, henceforth also referred to as “IMP”), xanthylic acid (xanthosine-5′-phosphate, henceforth also referred to as “XMP”), guanylic acid (guanosine-5′-monophosphate, henceforth also referred to as “GMP”), adenylic acid (adenosine-5′-monophosphate, henceforth also referred to as “AMP”), and so forth.
The phrase “ability to produce a purine-derived substance” or “is able to produce a purine-derived substance” means the ability of the Bacillus bacterium to produce, secrete, or cause accumulation of a purine-derived substance in the bacterial cells or the medium in which the bacterium is cultured to such an extent that the purine-derived substance can be collected from the cells or medium. The Bacillus bacterium may be able to produce two or more kinds of the aforementioned purine-derived substances.
The Bacillus bacterium which is able to produce a purine-derived substance may inherently have this ability, or may be modified as mentioned below to have this ability. Bacteria may be modified by using mutagenesis method or recombinant DNA techniques. Moreover, the Bacillus bacterium may be modified so that enzymatic activity of transaldolase is decreased, in such a manner as described later.
The phrase “enzymatic activity is decreased” or “to decrease the enzymatic activity” indicates that the enzymatic activity of transaldolase described above, or of an enzyme which decomposes a purine-derived substance such as inosine monophosphate (IMP) dehydrogenase is lower than that in an unmodified strain, for example, a wild-type strain of the Bacillus bacterium. This can also mean that the activity is substantially disappeared. The same shall apply to the activity of the purine operon repressor described later.
Examples of the Bacillus bacterium include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, and so forth.
Examples of Bacillus subtilis include Bacillus subtilis 168 Marburg (ATCC 6051), Bacillus subtilis PY79 (Plasmid, 1984, 12, 1-9) and so forth, and examples of Bacillus amyloliquefaciens include Bacillus amyloliquefaciens T (ATCC 23842), Bacillus amyloliquefaciens N (ATCC 23845), and so forth. Examples of Bacillus pumilus include Bacillus pumilus Gottheil No. 3218 (ATCC No. 21005, U.S. Pat. No. 3,616,206), and so forth. These strains can be obtained from American Type Culture Collection (Address: P.O. Box 1549, Manassas, Va. 20108, United States of America).
A Bacillus bacterium which is able to produce a purine-derived substance can be obtained, for example, by making the bacteria auxotrophic for purine nucleosides or resistant to purine analogues (Japanese Patent Publication Nos. 38-23099, 54-17033, 55-45199, 57-14160, 57-41915 and 59-42895). A Bacillus bacterium which is auxotrophic or drug resistant can be obtained by treating the bacterium with a known mutatgen such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or EMS (ethyl methanesulfonate).
Examples of Bacillus bacteria which produce a purine nucleoside include the following. A Bacillus strain which is able to produce inosine is Bacillus subtilis KMBS16. This strain is derived from the known Bacillus subtilis trpC2 strain (168 Marburg), by disrupting the following geens: purR encoding the purine operon repressor (purR::spc), purA encoding succinyl-AMP synthase (purA::erm), and deoD encoding purine nucleoside phosphorylase (deoD::kan) (Japanese Patent Laid-open No. 2004-242610, US2004166575A1). Bacillus subtilis AJ3772 strain (FERM P-2555, Japanese Patent Laid-open No. 62-014794) and so forth may also be used.
Examples of Bacillus bacteria which is able to produce guanosine include a Bacillus bacterium with increased IMP dehydrogenase activity (Japanese Patent Laid-open No. 3-58787), a Bacillus bacterium which is obtained by introducing a vector containing a gene conferring resistance to a purine analogue or decoyinine into an adenine auxotrophic mutant (Japanese Patent Publication No. 4-28357), and so forth.
Examples of Bacillus bacteria which produce a purine nucleotide include the following. Bacillus subtilis which have attenuated phosphatase activity have been reported to be able to produce inosinic acid (Uchida, K. et al., Agr. Biol. Chem., 1961, 25, 804-805; Fujimoto, M., Uchida, K., Agr. Biol. Chem., 1965, 29, 249-259). Examples of Bacillus bacteria which are able to produce guanylic acid, including 5′-guanylic acid (guanosine-5′-monophosphate, henceforth referred to as “GMP”), include mutants of Bacillus bacteria which are auxotrophic for adenine, and resistant to decoyinine or methionine sulfoxide (Japanese Patent Publication No. 56-12438).
Furthermore, bacteria which are able to produce xanthylic acid can be constructed by known methods for breeding coryneform bacteria, typically including Corynebacterium ammoniagenes. For example, a strain able to produce xanthylic acid can be obtained by enhancing PRPP amidotransferase (Japanese Patent Laid-open No. 8-168383), or making the strain resistant to aliphatic amino acids (Japanese Patent Laid-open No. 4-262790), or dehydroproline (South Korean Patent Unexamined Publication No. 2003-56490).
Moreover, another example of a method for breeding Bacillus bacteria which are able to produce purine-derived substances is to enhance the activities of enzymes involved in purine biosynthesis which are common to the biosynthesis of purine nucleosides and purine nucleotides, i.e., purine biosynthesis enzymes, in bacterial cells. The activity of the enzyme in the cells is preferably increased to a level greater than that of an unmodified strain of Bacillus bacterium, for example, a wild-type Bacillus bacterium. The phrase “activity is increased” includes, for example, when the number of enzyme molecules per cell is increased, and also when the specific activity per enzyme molecule is increased, and so forth. For example, the activity can be increased by increasing the expression of the gene which encodes the enzyme.
Examples of enzymes involved in purine biosynthesis include, for example, phosphoribosyl pyrophosphate amidotransferase, phosphoribosyl pyrophosphate synthetase (PRPP synthetase [EC: 2.7.6.1]), and so forth.
Some of the catabolites produced by the metabolism of sugar sources such as glucose that flow into the pentose phosphate pathway are converted into ribose-5-phosphate via ribulose-5-phosphate. From the biosynthesized ribose-5-phosphate, phosphoribosyl pyrophosphate (PRPP) is produced, which is an indispensable precursor for purine nucleoside, histidine and tryptophan biosyntheses. Specifically, ribose-5-phosphate is converted into PRPP by phosphoribosyl pyrophosphate synthetase. Therefore, the ability to produce purine-derived substances can be imparted to a Bacillus bacterium by modifying the bacterium so that the activity of phosphoribosyl pyrophosphate synthetase is increased.
The phrase “activity of phosphoribosyl pyrophosphate synthetase is increased” or “to increase phosphoribosyl pyrophosphate synthetase activity” means that the activity of phosphoribosyl pyrophosphate synthetase is increased as compared to that of an unmodified strain such as a wild-type strain or a parent strain. The activity of phosphoribosyl pyrophosphate synthetase can be measured by, for example, the method of Switzer et al. (Methods Enzymol., 1978, 51, 3-11) or Roth et al. (Methods Enzymol., 1978, 51, 12-17). A Bacillus bacterium in which the activity of phosphoribosyl pyrophosphate synthetase is increased can be obtained by, for example, increasing the expression of the gene encoding phosphoribosyl pyrophosphate synthetase in the Bacillus bacterium by introducing a plasmid containing the gene or integrating the gene into the chromosome (Japanese Patent Laid-open No. 2004-242610). Although the prs gene (SEQ ID NO: 3) derived from a Bacillus bacterium (Genbank Accession No. X16518) encodes phosphoribosyl pyrophosphate synthetase and may be used, genes derived from other bacteria, animals, or plants which encode a protein having phosphoribosyl pyrophosphate synthetase activity may also be used.
Furthermore, once PRPP is produced, some of it is converted into purine nucleotides and purine nucleosides by the enzymes involved in the purine biosynthesis. Examples of the genes encoding such enzymes include the genes of the purine operon from Bacillus subtilis, specifically, genes of the purEKB-purC(orf) QLF-purMNH(J)-purD operon (Ebbole D. J. and Zalkin H., J. Biol. Chem., 1987, 262, 17, 8274-87) (at present, also called purEKBCSQLFMNHD, Bacillus subtilis and Its Closest Relatives, Editor in Chief: A. L. Sonenshein, ASM Press, Washington D.C., 2002, Genbank Accession No. NC—000964), and the genes of the pur regulon from Escherichia coli (Escherichia and Salmonella, Second Edition, Editor in Chief: F. C. Neidhardt, ASM Press, Washington D.C., 1996).
Accordingly, enhancing expression of these genes imparts or enhances the ability to produce a purine-derived substance. In addition, genes of the purine operon which can be used are not limited to these, and genes derived from other microorganisms, animals, and plants may also be used.
Examples of the method for increasing the expression of the purine operon include increasing the expression of genes of the purine operon in a Bacillus bacterium by introducing a plasmid containing the genes or integrating the genes into the chromosome or the like.
The second method for increasing the expression of the purine operon is to replace the native promoter of the purine operon with a stronger promoter, and to replace the −35 or −10 region of the native promoter with a consensus sequence.
For example, in Bacillus subtilis (B. subtilis 168 Marburg strain, ATCC 6051), the −35 sequence of the purine operon is a consensus sequence (TTGACA), but the −10 sequence is TAAGAT, which differs from the consensus sequence TATAAT (Ebbole, D. J. and H. Zalikn, J. Biol. Chem., 1987, 262, 8274-8287). Therefore, by changing the −10 sequence (TAAGAT) to the similar consensus sequence TATAAT, TATGAT or TAAAAT, the transcriptional activity of the purine operon can be increased. The promoter sequence can be replaced by the same method as that of the gene substitution, which is described below.
The third method for increasing the expression of the purine operon is to reduce the expression of the purine operon repressor (U.S. Pat. No. 6,284,495). The phrase “expression of the purine operon repressor” includes both the transcription of the purine operon gene and the translation of the transcription product. Furthermore, “expression is decreased” means when the expression is lower than that in an unmodified strain such as a wild-type Bacillus bacterium, and also when the expression is substantially eliminated.
Expression of the purine operon repressor (purine repressor) can be decreased by, for example, irradiating the Bacillus bacterium with ultraviolet rays or treating the bacterium with a known mutagen such as NTG or EMS, and selecting a mutant with decreased expression of the purine repressor.
Furthermore, a Bacillus bacterium with decreased expression of the purine repressor can also be obtained by, for example, besides a mutagenesis treatment, replacing the gene encoding the purine repressor on the chromosome (purR, GenBank Accession NC—000964, coding region corresponds to the nucleotide numbers 54439 to 55293, SEQ ID NO: 5) with a corresponding gene that does not function normally (hereinafter, also referred to as a “disrupted-type gene”) by homologous recombination utilizing gene recombination techniques (Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press (1972); Matsuyama, S. and Mizushima, S., J. Bacteriol., 1985, 162, 1196-1202).
For example, the native gene can be replaced with a disrupted-type gene on the host chromosome in the manner as described below. Hereinafter, the disruption of the purR gene is described. Other genes such as purA, deoD, guaB, fbp and ywjH genes can be similarly disrupted.
A plasmid which is not capable of replicating in the chosen host, such as Bacillusbacteria, or the like, is constructed to have a sequence which is homologous to a sequence on the chromosome of the Bacillus bacteria. When this plasmid is introduced into the bacterial cell, recombination at the site of the homologous sequence occurs at a certain frequency. The entire plasmid is then recombined into the chromosome. Thereafter, if further recombination occurs at the site of the homologous sequence, the plasmid is deleted from the chromosome. At this time, depending on the site where the recombination occurs, the disrupted-type gene may remain on the chromosome, and the original native gene is deleted from the chromosome with the plasmid. By this method, a strain in which the native purR gene on the chromosome is replaced with the disrupted-type purR gene is obtained.
Disrupting genes using homologous recombination techniques is well-known, and includes when linear DNA and/or a temperature-sensitive plasmid is/are used, and so forth. Furthermore, the purR gene can also be disrupted by using a plasmid containing the purR gene and a marker gene, such as a drug resistance gene, and which is not able to replicate in the target bacterial cell. That is, in a cell that has been transformed with such a plasmid, the marker gene is incorporated into the chromosomal DNA and imparts drug resistance. Since the marker gene is incorporated into the chromosome at a high rate by homologous recombination of the purR gene sequences that sandwich the marker gene on the plasmid with the purR gene on the chromosome, bacterial strains containing the disrupted purR gene can be selected efficiently.
The disrupted-type purR gene used for the gene disruption can be obtained by, specifically, deleting a particular region of the purR gene by digestion with a restriction enzyme and re-ligation, inserting another DNA fragment (marker gene etc.) into the purR gene, or substituting, deleting, inserting, adding, or inverting one or more nucleotides in the nucleotide sequence of the coding region, promoter region, or the like of the purR gene by site-specific mutagenesis (Kramer, W. and Frits, H. J., Methods in Enzymology, 1987, 154, 350-367) recombinant PCR (PCR Technology, Stockton Press (1989)) or treatment with a chemical agent such as sodium hyposulfite or hydroxylamine (Shortle, D. and Nathans, D., Proc. Natl. Acad. Sci. U.S.A., 1978, 75, 2170-2174). Then, the strain with decreased purR repressor activity or decreased purR gene transcription can be selected. Among these methods, either deleting a particular region of the purR gene by digestion with a restriction enzyme and re-ligation, or inserting another DNA fragment into the purR gene, is preferable in view of reliability and stability. The particular region of the purR gene to be deleted may be a 5′ end sequence, internal sequence, or 3′ end sequence. However, if the region includes 90% or more, more preferably 95% or more, particularly 97% or more, of the full length purR gene, it is more likely to ensure a reduction in repressor activity. Furthermore, when a frame shift mutation is caused by deletion or insertion of nucleotides in the coding region of the purR gene, it is preferable to delete or insert nucleotides at multiple sites on the 3′ end, so as to ensure reduction of the repressor activity.
The purine repressor activity can also be reduced by, besides the aforementioned gene disruption, using well-known mutagenesis methods to introduce a mutation that reduces the intracellular purine repressor activity into the purR gene on the chromosome. For example, an amino acid substitution (missense mutation), a stop codon (nonsense mutation), or a frame shift mutation that adds or deletes one or two nucleotides can be introduced, or the gene can be partially or entirely deleted. Furthermore, the activity of the repressor can also be decreased by inserting a transposon into the purR gene on the chromosome.
The activity of the purine repressor can also be reduced by replacing an expression control sequence of the purR gene, such as promoter, on the chromosomal DNA with a weaker one. The strength of a promoter is defined by the frequency of initiation of RNA synthesis. Examples of methods for evaluating the strength of promoters and strong promoters are described in the paper of Goldstein et al. (Prokaryotic promoters in biotechnology, Biotechnol. Annu. Rev., 1995, 1, 105-128), and so forth. Furthermore, several nucleotides in the promoter region of the target gene can be substituted with a nucleotide, resulting in a weaker promoter (International Patent Publication WO00/18935). Furthermore, it is known that several nucleotides in the spacer region between the ribosome binding site (RBS) and the start codon can be substituted, in particular, the sequence immediately upstream from the start codon, and as a result, the translation efficiency of the mRNA is greatly effected. This modification of the RBS may be combined with decreasing the transcription of the target gene.
Furthermore, a recombinant DNA may be prepared which contains a mutation that destabilizes the purR messenger RNA. This DNA can then be transformed into a host Bacillus bacterium.
The activities of the enzymes encoded by the purA, deoD, guaB, fbp and ywjH genes described later can also be decreased in the same manner as described above.
The purR gene can be obtained from the chromosomal DNA of a microorganism which contains the purine operon by PCR using oligonucleotide primers prepared based on the known nucleotide sequence of the purR gene. The purR gene can also be obtained from a chromosomal DNA library of a microorganism which contains the purine operon by hybridization using an oligonucleotide probe prepared on the basis of the known nucleotide sequence of the purR gene. The nucleotide sequence of the purR gene from the Bacillus subtilis 168 Marburg strain has been reported [GenBank accession No. D26185 (the coding region corresponds to the nucleotide numbers 118041 to 118898), or NC—000964 (the coding region corresponds to the nucleotide numbers 54439 to 55296)). The nucleotide sequence of the purR gene and the amino acid sequence encoded by the gene are shown in SEQ ID NOS: 5 and 6, respectively (Japanese Patent Laid-open No. 2004-242610).
Primers used to obtain the purR gene in PCR may be any primer which allows for amplification of a part or the full length of the purR gene, and specific examples include oligonucleotides having the nucleotide sequences shown in SEQ ID NO: 17 (GAAGTTGATGATCAAAA) and SEQ ID NO: 18 (ACATATTGTTGACGATAAT).
The purR gene used for preparation of the disrupted-type gene does not necessarily need to be the full length purR gene, but should be of sufficient length to cause gene disruption. Moreover, the microorganism used to obtain each gene is not particularly limited, so long the gene native to the chosen microorganism is sufficiently homologous to the purR gene of the Bacillus bacterium to cause homologous recombination. However, it is usually preferable to use the gene which is native to the Bacillus bacterium when chosen as the host.
Examples of the marker gene include drug resistance genes such as the spectinomycin resistance, kanamycin resistance, and tetracycline resistance genes. The spectinomycin resistance gene of Enterococcus faecalis can be obtained by preparing the pDG1726 plasmid from Escherichia coli ECE101, which is commercially available from the Bacillus Genetic Stock Center (BGSC), and removing the resistance gene as a cassette from the plasmid. The erythromycin resistance gene of Staphylococcus aureus can be obtained by preparing the pDG646 plasmid from Escherichia coli ECE91, which is commercially available from the Bacillus Genetic Stock Center (BGSC), and removing the resistance gene as a cassette from the plasmid. The kanamycin resistance gene derived from Streptococcus faecalis can be obtained by preparing the pDG783 plasmid from Escherichia coli ECE94, which is commercially available from the Bacillus Genetic Stock Center (BGSC), and removing the resistance gene as a cassette from the plasmid. Furthermore, the chloramphenicol resistance gene from Staphylococcus aureus can be obtained by preparing the pC194 plasmid from Bacillus subtilis 1E17, which is commercially available from the Bacillus Genetic Stock Center (BGSC), and amplifying the plasmid by PCR the plasmid as the template. The tetracycline resistance gene of Streptococcus agalactiae can be obtained by preparing the pDG1513 plasmid from Escherichia coli ECE99, and removing the plasmid as a cassette from the plasmid (Gene, 1995, 167:335-336).
When a drug resistance gene is used as the marker gene, a strain with a disrupted purR gene can be obtained by inserting the drug resistance gene into the purR gene on a plasmid at an appropriate site, transforming the chosen microorganism with the plasmid, and selecting a drug-resistant transformant. Disruption of the purR gene on the chromosome can be confirmed by Southern blotting or PCR. Incorporation of the aforementioned spectinomycin resistance gene, erythromycin resistance gene, or kanamycin resistance gene into the chromosomal DNA can be confirmed by PCR using primers which can amplify these genes.
Expression of the purine operon is regulated by the terminator-antiterminator sequence located downstream of the promoter (henceforth referred to as the attenuator sequence) (Ebbole, D. J. and Zalkin, H., J. Biol. Chem., 1987, 262, 8274-8287; Ebbole D. J. and Zalkin H., J. Biol. Chem., 1988, 263, 10894-10902; Ebbole, D. J. and Zalkin, H., J. Bacteriol., 1989, 171, 2136-2141). Therefore, expression of the purine operon can be increased by deleting the attenuator sequence. The attenuator sequence can be deleted by the same method as for the disruption of purR.
In order to further increase transcription of the purine operon, any of the methods described above may be combined. For example, the purR gene may be disrupted, and further, the purine operon without the attenuator sequence may be amplified using a plasmid, or multiple copies of this modified purine operon may be introduced into the chromosome.
The activities of enzymes involved in purine biosynthesis can also be enhanced by desensitizing enzymes which negatively regulate purine biosynthesis, for example, by desensitizing the enzymes which regulate feedback inhibition (WO99/03988).
Furthermore, the ability to produce purine-derived substances can also be enhanced by attenuating the uptake of the purine-derived substances into the cells. For example, the uptake of purine nucleosides by the cells can be attenuated by blocking a reaction which facilitates this uptake. Examples of reactions involved in the uptake of the purine nucleosides by the cells include reactions which are catalyzed by nucleoside permeases.
Furthermore, when a purine nucleoside is produced, enzymes which act to decompose the purine nucleoside may be decreased, which will result in increased production of the purine nucleoside. An example of such an enzyme is purine nucleoside phosphorylase.
Purine nucleotides which are synthesized from PRPP by enzymes involved in purine biosynthesis are dephosphorylated and thereby converted into a purine nucleoside. To efficiently produce a purine nucleoside, it is preferable to reduce the activity of purine nucleoside phosphorylases, which further degrade purine nucleosides into hypoxanthine or the like. That is, it is preferable to attenuate or eliminate the activity of the purine nucleoside phosphorylase that uses purine nucleosides, such as inosine, as a substrate.
Specifically, the purine nucleoside phosphorylase activity can be decreased by disrupting the deoD and pupG genes in Bacillus bacteria. The Bacillus bacterium may be modified by disrupting one or both of the deoD and pupG genes. The deoD and the pupG genes, for example, derived from or native to Bacillus bacteria (deoD: Genbank Accession No. NC—000964 (SEQ ID NO: 7), pupG: Genbank Accession No. NC—000964 (SEQ ID NO: 9)) can be used, and the gene-disrupted strain can be obtained in the same manner as that described for the aforementioned disruption of the purR gene.
The ability to produce a purine-derived substance may also be enhanced by decreasing the activity of succinyl-AMP synthase. An example of the gene encoding succinyl-AMP synthase is the purA gene. An example of the purA gene is the gene having the nucleotide sequence registered as GenBank Accession No. NC—000964 (coding region corresponds to the nucleotide numbers 4153460 to 4155749 of the complementary strand, SEQ ID NO: 11).
The ability to produce a purine-derived substance may also be enhanced by decreasing the activity of inosine monophosphate (IMP) dehydrogenase. An example of the gene encoding IMP dehydrogenase is the guaB gene. An example of the guaB gene is the gene having the nucleotide sequence registered as GenBank Accession No. NC—000964 (coding region corresponds to the nucleotide numbers 15913 to 17376, SEQ ID NO: 13).
Furthermore, the ability to produce a purine-derived substance may also be enhanced by decreasing the activity of fructose bisphosphatase. An example of the gene encoding fructose bisphosphatase is the fbp gene. An example of the fbp gene is the gene having the nucleotide sequence registered as GenBank Accession No. NC—000964 (coding region corresponds to the nucleotide numbers 4127053 to 4129065, SEQ ID NO: 15).
Moreover, genes which encode proteins which act to enhance secretion of a purine-derived substance may be overexpressed in the method to increase the ability of a microorganism to produce purine-derived substances. An example of a bacterium in which such a gene has been overexpressed is a Bacillus bacterium in which the rhtA gene is overexpressed (Japanese Patent Laid-open No. 2003-219876).
The purR, deoD, pupG, purA, guaB, and fbp genes to be disrupted as described above, and the prs gene, which is to be overexpressed, may include conservative variants, for example, DNAs encoding proteins having the amino acid sequences of SEQ ID NOS: 6, 8, 10, 12, 14, 16 and 4, respectively, but which may contain substitutions, deletions, insertions, additions, or inversions of one or several amino acid residues and yet still maintain their native activity, that is, the activities of the purine repressor, purine nucleoside phosphorylase, succinyl-AMP synthase, IMP dehydrogenase, fructose bisphosphatase or phosphoribosyl pyrophosphate synthetase, respectively. The number of amino acids to be changed may be, for example, 1 to 50, preferably 1 to 30, more preferably 1 to 10.
These changes in the amino acid sequences as described above are usually conservative changes so that the native activities are maintained. Examples of conservative amino acid substitutions include: substitution of Ser or Thr for Ala; substitution of Gln, His or Lys for Arg; substitution of Glu, Gln, Lys, His or Asp for Asn; substitution of Asn, Glu or Gln for Asp; substitution of Ser or Ala for Cys; substitution of Asn, Glu, Lys, His, Asp or Arg for Gln; substitution of Asn, Gln, Lys or Asp for Glu; substitution of Pro for Gly; substitution of Asn, Lys, Gln, Arg or Tyr for His; substitution of Leu, Met, Val or Phe for Ile; substitution of Ile, Met, Val or Phe for Leu; substitution of Asn, Glu, Gln, His or Arg for Lys; substitution of Ile, Leu, Val or Phe for Met; substitution of Trp, Tyr, Met, Ile or Leu for Phe; substitution of Thr or Ala for Ser; substitution of Ser or Ala for Thr; substitution of Phe or Tyr for Trp; substitution of His, Phe or Trp for Tyr; and substitution of Met, Ile or Leu for Val.
Specific examples of conservative variants of the purR, deoD, pupG, purA, guaB, and fbp genes and the prs gene described above include DNAs which are homologous, for example, 70% or more, preferably 80% or more, more preferably 90% or more, particularly preferably 95% or more, to DNAs having the nucleotide sequences of SEQ ID NOS: 5, 7, 9, 11, 13, 15 and 3, respectively. More specifically, the examples of the conservative variants include DNAs that are able to hybridize with DNAs having nucleotide sequences complementary to the nucleotide sequences of SEQ ID NOS: 5, 7, 9, 11, 13, 15 and 3 under stringent conditions. An example of the stringent conditions is washing at 60° C. and salt concentrations of 1×SSC, 0.1% SDS, preferably 0.1×SSC, 0.1% SDS, one or more times, preferably two or three times.
Homology of DNAs can be evaluated by a BLAST or FASTA search, the calculation method of Crustal W, and so forth.
BLAST (basic local alignment search tool) is a heuristic search algorithm used by the programs blastp, blastn, blastx, megablast, tblastn and tblastx, and the results obtained by these programs are considered significant on the basis of the statistical method of Karlin, Samuel, and Stephen F. Altschul (“Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes”, Proc. Natl. Acad. Sci. USA, 1990, 87:2264-68; “Applications and statistics for multiple high-scoring segments in molecular sequences”, Proc. Natl. Acad. Sci. USA, 1993, 90:5873-7). The FASTA search method was described by W. R. Pearson (“Rapid and Sensitive Sequence Comparison with FASTP and FASTA”, Methods in Enzymology, 1990 183:63-98). The Clustal W method is described by Thompson J. D., Higgins D. G., and Gibson T. J. (“CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice”, Nucleic Acids Res., 1994, 22:4673-4680).
Moreover, DNA used to prepare the disrupted-type gene may also be conservative variants of the purR, deoD, pupG, purA, or guaB genes.
The target gene may be incorporated into the chromosomal DNA of Bacillus bacterium in the same manner as that for the gene encoding transaldolase described later.
(2) The Modification for Decreasing the Enzymatic Activity of Transaldolase
The Bacillus bacterium can be obtained by modifying a strain having the ability to produce a purine-derived substance such as those described above so that the enzymatic activity of transaldolase is decreased. The order of modification is not limited, and after modifying the bacterium so that the enzymatic activity of transaldolase is decreased, the ability to produce purine nucleotides may be imparted to the bacterium.
Transaldolase is an enzyme which catalyzes the reaction of reversibly generating D-erythrose-4-phosphate and D-fructose-6-phosphate from sedoheptulose-7-phosphate and D-glyceraldehyde-3-phosphate, which is one of the reactions of the pentose phosphate pathway. The “pentose phosphate pathway” means the pathway in which glucose taken up into cells is phosphorylated by glucose kinase to biosynthesize glucose-6-phosphate, then glucose-6-phosphate is oxidatively converted into ribose-5-phosphate, and the pathway of the reversible interconversion of phosphates of triose, tetrolose, pentose, hexose, and heptose by the actions of epimerase, transketolase (EC: 2.2.1.1), and transaldolase (EC: 2.2.1.2).
The enzymatic activity of transaldolase can be measured by the following method. For example, the activity can be measured by converting the generated D-glyceraldehyde-3-phosphate into hydroxyacetone phosphate with triosephosphate isomerase and measuring hydroxyacetone phosphate using glycerol-3-phosphate dehydrogenase and NADH (Ochoa, T., and Horecker, B. L., 1966, Methods Enzymol., 9, 499-505).
The modification which results in a decrease of the enzymatic activity of transaldolase can be attained by, for example, as explained above for the disruption of the purR gene. That is, the enzymatic activity can be decreased by substituting a corresponding gene which does not function normally (e.g., a disrupted-type gene obtained by inserting a marker gene such as a drug resistance gene into the transaldolase gene) for the transaldolase gene on the chromosome by homologous recombination. Furthermore, as described for the purR gene, mutations which result in reducing the intracellular enzymatic activity of transaldolase may be introduced into the transaldolase gene on the chromosome by conventional mutagenesis methods.
Examples of transaldolase from Bacillus subtilis include the protein having 212 amino acid residues as shown in SEQ ID NO: 2, and the gene encoding the protein, preferably the gene having the nucleotide sequence of SEQ ID NO: 1 (ywjH gene, Genbank Accession No. NC—000964), can be used in the above described modification procedures. The ywjH gene is located at about 325° on the Bacillus subtilis chromosome.
The gene encoding transaldolase may also be a conservative variant of the ywjH gene, like the aforementioned genes. Specifically, examples include a DNA encoding a protein having the amino acid sequence of SEQ ID NO: 2, but which includes substitutions, deletions, insertions, additions, or inversions of one or several amino acid residues while maintaining the enzymatic activity of transaldolase. Examples further include a DNA encoding a protein having a homology of 50% or more, preferably 70% or more, more preferably 80% more, particularly preferably 90% or more, most preferably 95% or more, to the amino acid sequence shown in SEQ ID NO: 2, and having the enzymatic activity of transaldolase. More specifically, examples include a DNA which is able to hybridize with the DNA having the nucleotide sequence of SEQ ID NO: 1 under stringent conditions. The stringent conditions include washing at 60° C. and salt concentrations of 1×SSC, 0.1% SDS, preferably 60° C., 0.1×SSC, 0.1% SDS, one or more times, preferably two or three times.
The DNA encoding a protein substantially identical to transaldolase as described above can be obtained, for example, by modifying the nucleotide sequence encoding such an enzyme so that an amino acid residue in a specific portion is substituted, deleted, inserted, added, or inverted by site-specific mutagenesis. Such a modified DNA as described above may also be obtained by a conventionally known mutagenesis treatment, such as in vitro treatment of DNA before the mutagenesis treatment with hydroxylamine, and treatment of a microorganism such as an Escherichia bacterium containing the DNA before the mutagenesis treatment with ultraviolet irradiation or a known mutagen such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid.
The target gene can be obtained by, for example, PCR (polymerase chain reaction, White, T. J. et al., Trends Genet., 1989, 5, 185-189) using a chromosomal DNA of a Bacillus bacterium as the template and oligonucleotide primers prepared based on the nucleotide sequence of the target gene. The chromosomal DNA can be prepared from a bacterium serving as a DNA donor by, for example, the method of Saito and Miura (H. Saito and K. Miura, Biochem. Biophys. Acta, 1963, 72, 619-629; Text for Bioengineering Experiments, Edited by the Society for Bioscience and Bioengineering, Japan, pp. 97-98, Baifukan, 1992) or the like. The primers for PCR can be prepared based on the known gene sequence from a Bacillus bacterium, or based on a conserved region among known genes from other bacteria.
Examples of the vector which can be used to incorporate the target gene into the chromosomal DNA of Bacillus bacterium include a vector having a temperature sensitive replication origin, such as pHV1248 (Prtit, M.-A., et. al., J. Bacteriol., 1990, 172, 6736-6740), vectors for E. coli such as pHSG398 (Takara Shuzo) and pBluescript SK-(Stratagene), and so forth.
In order to ligate the target gene to a vector carrying a marker which functions in Bacillus bacteria, the vector is digested with a restriction enzyme which generates sticky ends compatible with the objective gene. The ligation is usually performed with a ligase such as T4 DNA ligase.
To introduce the recombinant DNA vector prepared as described above into a Bacillusbacterium, any known transformation method can be employed. Examples include, for instance, preparing competent cells from cells which are at the growth phase followed by introducing the DNA thereinto (Dubunau D. and Davidoff-Abelson, R., J. Mol. Biol., 1971, 56, 209-221), and making host cells into protoplasts or spheroplasts, which can easily take up recombinant DNA, followed by introducing the recombinant DNA into the DNA-acceptor cells (Chang, S. and Choen, S. N., Molec. Gen. Genet., 1979, 168, 111-115).
<2> Method for Producing a Purine-Derived Substance
The Bacillus bacterium prepared as described above efficiently produces a purine-derived substance. Therefore, by culturing the Bacillus bacterium as described above in an appropriate medium, a purine-derived substance, such as a purine nucleoside and a purine nucleotide, can be produced and will accumulate in the bacterial cells or the medium.
The medium used in the culture can be any conventional medium which contains a carbon source, nitrogen source, and mineral salts, as well as organic trace nutrients such as amino acids and vitamins, as required. Either a synthetic or natural medium may be used. Any carbon source and nitrogen source may be used so long as they can be utilized by the chosen strain.
As the carbon source, sugars such as glucose, fructose, sucrose, maltose, mannose, galactose, arabinose, xylose, trehalose, ribose, starch hydrolysates and molasses, and alcohols such as glycerol and mannitol can be used, and organic acids such as gluconic acid, acetic acid, citric acid, maleic acid, fumaric acid and succinic acid can also be used independently or in combination with other carbon sources.
As the nitrogen source, ammonia, ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate and ammonium acetate, nitric acid salts, organic nitrogen such as soybean hydrolysate, and so forth can be used.
As the organic trace nutrients, amino acids, vitamins, fatty acids, nucleic acids, and substances containing these, such as peptone, casamino acid, yeast extract and soybean protein decomposition product and so forth can be used. When an auxotrophic mutant strain that requires an amino acid or the like for growth is used, it is necessary to supplement the required nutrient.
As the mineral salts, phosphoric acid salts, magnesium salts, calcium salts, iron salts, manganese salts and so forth can be used.
Although the culture conditions may vary depending on the type of Bacillus bacterium, Bacillus subtilis, for example, is cultured as an aeration culture, while the fermentation temperature is controlled to 20 to 50° C., and pH to 4 to 9. When pH falls during the culture, the medium is neutralized with an alkali such as ammonia gas. A purine nucleoside is produced into the medium after about 40 hours to 3 days of culture in such a manner as described above.
After completion of the culture, the purine-derived substance which has accumulated in the medium may be collected in a conventional manner. For example, it can be isolated by precipitation, ion exchange chromatography, and so forth.
Furthermore, if the chosen microorganism lacks a gene encoding a nucleosidase or nucleotidase, a corresponding nucleoside or nucleotide can be produced. Furthermore, if inosine auxotrophy is imparted, a precursor or relevant substances involved in the biosynthesis pathway thereof can be produced.
Furthermore, by reacting inosine or guanosine prepared by the described method with purine nucleoside phosphorylase or phosphoribosyltransferase, 5′-inosinic acid or 5′-guanylic acid can be obtained.
Moreover, it is also possible to phosphorylate the purine nucleoside produced using the microorganism as described herein by reacting phosphotransferase with the purine nucleoside to produce a purine nucleotide (nucleoside 5′-phosphoric acid ester) (Japanese Patent Laid-open No. 2000-295996). For example, the method for producing a purine nucleotide using an Escherichia bacterium transformed with the gene encoding inosine guanosine kinase of Escherichia coli (WO91/08286), and the method for producing a purine nucleotide using Corynebacterium ammoniagenes transformed with the gene encoding inosine guanosine kinase of Exiguobacterium acetylicum is introduced (WO96/30501) can be used.
Moreover, it is also possible to produce a purine nucleotide (nucleoside 5′-phosphoric acid ester) by reacting the purine nucleoside produced by the microorganism as described herein with a phosphate donor such as polyphosphoric acid, phenyl phosphate, and carbamyl phosphate, and a microorganism which is able to produce a nucleoside 5′-phosphoric acid ester or acid phosphatase (EC 3.1.3.2). Although the microorganism which is able to produce a nucleoside 5′-phosphoric acid ester is not particularly limited so long as it can convert a purine nucleoside into a purine nucleotide, examples include, for example, the microorganism disclosed in International Patent Publication WO96/37603.
Moreover, Escherichia blattae JCM 1650, Serratia ficaria ATCC 33105, Klebsiella planticola IFO 14939 (ATCC 33531), Klebsiella pneumoniae IFO 3318 (ATCC 8724), Klebsiella terrigena IFO 14941 (ATCC 33257), Morganella morganii IFO 3168, Enterobacter aerogenes IFO 12010, Enterobacter aerogenes IFO 13534 (ATCC 13048), Chromobacterium fluviatile IAM 13652, Chromobacterium violaceum IFO 12614, Cedecea lapagei JCM 1684, Cedecea davisiae JCM 1685, Cedecea neteri JCM 5909, and so forth disclosed in Japanese Patent Laid-open No. 07-231793 can also be used.
The acid phosphatase, for example, disclosed in Japanese Patent Laid-open No.2002-000289 can be used. The acid phosphatase with increased affinity to a nucleoside (Japanese Patent Laid-open No. 10-201481), a mutant acid phosphatase with decreased nucleotidase activity (WO96/37603), a mutant acid phosphatase with decreased phosphoric acid ester hydrolysis activity (Japanese Patent Laid-open No. 2001-245676) and so forth can be more preferably be used.
It is also possible to obtain a purine nucleotide by chemically phosphorylating the purine nucleoside produced using the microorganism (Bulletin of the Chemical Society of Japan, 42, 3505). Moreover, the method of obtaining GMP by coupling the microorganism with the ability to produce XMP and XMP aminase activity using the ATP-regenerating system of the microorganism, and the method of obtaining IMP by coupling inosine kinase (Biosci. Biotech. Biochem., 51, 840 (1997); Japanese Patent Laid-open No. 63-230094) can also be used.
The inosine, guanosine, or purine nucleoside prepared by the above-described method may be purified, or may be a purine nucleoside fermentation broth, or a crude product containing a purine nucleoside.
EXAMPLES
Hereafter, the present invention will be more specifically explained with reference to the following non-limiting examples.
Example 1
Construction of a Bacterial Strain Deficient in the pupG and deoD Genes
A strain deficient in the purine nucleoside phosphorylase gene (deoD) was constructed from the recombinant strain KMBS310 as described below. The KMBS310 strain (Japanese Patent Application No. 2005-280186), which is derived from Bacillus subtilis (B. subtilis 168 Marburg strain, ATCC 6051), is deficient in the purine operon repressor gene (purR), succinyl-AMP synthase gene (purA) and purine nucleoside phosphorylase gene (pupG), and has attenuated IMP dehydrogenase gene (guaB), (Japanese Patent Application No. 2005-280186). In this strain, expression of the purine operon and the PRPP synthetase gene was enhanced by modifying the promoter region and the SD sequence, respectively.
Genomic DNA was prepared from the KMBS16 strain (purR::spc purA::erm deoD::kan, Japanese Patent Laid-open No. 2004-242610) by the method of Fouet and Sonenshein (J. Bacteriol., 1990, 172, 835-844), and was used to transform competent cells of the B. subtilis 168 Marburg strain prepared by the method of Dubnau and Davidoff-Abelson (J. Mol. Biol., 1971, 56, 209-221), and colonies grew on an LB agar plate containing 5 μg/ml of kanamycin. The colonies which did not become spectinomycin-resistant nor erythromycin-resistant were selected, and one strain among them was designated KMBS5 (deoD::kan).
Genomic DNA was prepared from KMBS5 by the method of Fouet and Sonenshein (J. Bacteriol., 1990, 172, 835-844), and was used to transform competent cells of the KMBS310 strain prepared by the method of Dubnau and Davidoff-Abelson (J. Mol. Biol., 1971, 56, 209-221), and colonies grew on an LB agar plate containing 5 μg/ml of kanamycin and 20 μg/ml of guanine. Several colonies were selected as described above, and one of the transformants was confirmed to have the deoD::kan substituted for the wild-type deoD gene, and all the mutations derived from KMBS310 were not replaced with wild-type sequences. This strain was designated KMBS321.
Example 2
Construction of a Bacterial Strain Deficient in Either the fbp Gene Encoding fructose bisphosphatase, or the ywjH Gene Encoding transaldolase, or Both, and Culture and Evaluation Thereof
(1) Preparation of the fbp Gene-Deficient Strain
A strain deficient in the fructose bisphosphatase gene (fbp) was constructed from the aforementioned recombinant strain KMBS321 as described below. The KMBS321 strain, which is derived from Bacillus subtilis (B. subtilis 168 Marburg strain, ATCC 6051), is deficient in the purine operon repressor gene (purR), succinyl-AMP synthase gene (purA) and purine nucleoside phosphorylase gene (pupG), and has attenuated IMP dehydrogenase gene (guaB), and has a modified purine operon promoter region and SD sequence of the PRPP synthetase gene (prs).
(i) Amplification of fbp Upstream Region by PCR
28-mer and 50-mer PCR primers having the following nucleotide sequences were prepared based on the information from the public gene data bank (GenBank Accession Nos. NC—000964 and V01277).
TTCCCTTAGGGTTATTTTCGTTTCAAAA (SEQ ID NO: 19) cgtttgttgaactaatgggtgctTTTATGAGCATGTGCATGATAAGGTGA (SEQ ID NO: 20, the nucleotides indicated with small letters correspond to the promoter upstream region of the chloramphenicol resistance gene (cat) cloned in pC194)
PCR (98° C. for 10 second, 55° C. for 30 seconds, 72° C. for 1.5 minutes, 30 cycles, Gene Amp PCR System Model 9600, Perkin-Elmer) was performed using the chromosomal DNA of the B. subtilis 168 Marburg strain as the template and the aforementioned primers to obtain an amplification fragment containing the fbp gene 5′ end region and about 1350 bp of the upstream region.
(ii) Amplification of fbp Downstream Region by PCR
50-mer and 27-mer PCR primers having the following nucleotide sequences were prepared based on the information from the public gene data bank (GenBank Accession Nos. NC—000964 and V01277).
acagctccagatccatatccttcttTTTTAGAGAGTTTGCGGGAGTATCG (SEQ ID NO: 21, the nucleotides indicated with small letters correspond to a downstream region of structural gene of the chloramphenicol resistance gene (cat) cloned in pC194)
TAAAGGTTTTTCGGGATAAGATTGAAA (SEQ ID NO: 22)
PCR (98° C. for 10 second, 55° C. for 30 seconds, 72° C. for 1.5 minutes, 30 cycles, Gene Amp PCR System Model 9600, Perkin-Elmer) was performed using the chromosomal DNA of the B. subtilis 168 Marburg strain as the template and the aforementioned primers to obtain an amplification fragment containing the fbp gene 3′ end region and about 1770 bp of the downstream region.
(iii) Amplification of cat Gene by PCR
50-mer PCR primers having the following nucleotide sequences were prepared based on the information from the public gene data bank (GenBank Accession Nos. V01277 and NC—000964).
tcaccttatcatgcacatgctcataaaAGCACCCATTAGTTCAACAAACG (SEQ ID NO: 23, the nucleotides indicated with small letters correspond to the sequence of 5′ end region of the fbp gene, and they were designed so as to be complementary to the 3′ end region of SEQ ID NO: 20)
cgatactcccgcaaactctctaaaaAAGAAGGATATGGATCTGGAGCTGT (SEQ ID NO: 24, the nucleotides indicated with small letters correspond to the sequence of 3′ end region of the fbp gene, and they were designed so as to be complementary to the 3′ end region of SEQ ID NO: 21)
PCR (98° C. for 10 second, 55° C. for 30 seconds, 72° C. for 1.5 minutes, 30 cycles, Gene Amp PCR System Model 9600, Perkin-Elmer) was performed by using the plasmid pC194 carrying the chloramphenicol resistance gene (cat) as the template and the aforementioned primers to obtain an amplification fragment of about 980 bp containing the cat gene.
(iv) Amplification of Fragment Comprising fbp Region Inserted with cat Gene by Recombinant PCR
The DNA fragments amplified in (i) to (iii) as described above were purified using MicroSpin Column S-400 (Amersham Pharmacia Biotech), and then a mixture of these primers in appropriate amounts was used as the template together with nucleotides having the sequences of SEQ ID NOS: 19 and 22 to perform PCR (98° C. for 10 second, 55° C. for 30 seconds, 72° C. for 4.5 minutes, 30 cycles, Gene Amp PCR System Model 9600, Perkin-Elmer) and thereby obtain a fragment containing the fbp region with the cat gene inserted therein.
(v) Preparation of fbp-Disrupted Inosine-Producing Strain
The DNA fragment including the fbp region into which the cat gene (fbp::cat) obtained in (iv) had been inserted was subjected to agarose gel electrophoresis, and the target fragment was extracted from the gel. The DNA fragment purified as described above was used to transform competent cells of the B. subtilis KMBS321 strain prepared by the method of Dubnau and Davidoff-Abelson (J. Mol. Biol., 1971, 56, 209-221), and colonies grew on an LB agar plate containing 2.5 μg/ml of chloramphenicol and 20 μg/ml of guanine. Chromosomal DNAs were prepared from these colonies, strains in which fbp region on the chromosome was replaced with the fbp region of which internal sequence was replaced with the chloramphenicol resistance gene (fbp::cat) by double recombination were identified by the PCR method described in (iv), and one of these strains was designated TABS133.
(2) Preparation of ywjH Gene-Deficient Strain
A strain deficient in the transaldolase gene (ywjH) was constructed from the aforementioned recombinant strain KMBS321 as described below. The KMBS321 strain (Japanese Patent Application No. 2005-280186), which is derived from Bacillus subtilis (B. subtilis 168 Marburg strain, ATCC 6051), is deficient in the purine operon repressor gene (purR), succinyl-AMP synthase gene (purA) and purine nucleoside phosphorylase gene (pupG). This strain also has an attenuated IMP dehydrogenase gene (guaB), wherein the purine operon promoter region and SD sequence of the PRPP synthetase gene (prs) are modified, the strain is deficient in the purine nucleoside phosphorylase gene (deoD).
(i) Amplification of ywjH Upstream Region by PCR
28-mer and 50-mer PCR primers having the following nucleotide sequences were prepared based on the information from the public gene data bank (GenBank Accession Nos. NC—000964 and V01277).
ATGGACGGAATCGAAATCTTAAAACGGA (SEQ ID NO: 25) cgtttgttgaactaatgggtgctttAGAATTCCTAATTCATTCGCTTCTC (SEQ ID NO: 26, the nucleotides indicated with small letters correspond to the promoter upstream region of the chloramphenicol resistance gene (cat) cloned in pC194).
PCR (98° C. for 10 second, 55° C. for 30 seconds, 72° C. for 1.5 minutes, 30 cycles, Gene Amp PCR System Model 9600, Perkin-Elmer) was performed using the chromosomal DNA of the B. subtilis 168 Marburg strain as the template and the aforementioned primers to obtain an amplification fragment containing the ywjH gene 5′ end region and about 1420 bp upstream region.
(ii) Amplification of the ywjH Downstream Region by PCR
50-mer and 28-mer PCR primers having the following nucleotide sequences were prepared based on the information from the public gene data bank (GenBank Accession Nos. NC—000964 and V01277).
acagctccagatccatatccttctTTGACCTGATTTCAGAAGTTAAACAG (SEQ ID NO: 27, the nucleotides indicated with small letters correspond to a downstream region of structural gene of the chloramphenicol resistance gene (cat) cloned in pC194)
GGATGACGTTATCGATAATGACTTCCTT (SEQ ID NO: 28)
PCR (98° C. for 10 second, 55° C. for 30 seconds, 72° C. for 1.5 minutes, 30 cycles, Gene Amp PCR System Model 9600, Perkin-Elmer) was performed using the chromosomal DNA of the B. subtilis 168 Marburg strain as the template and the aforementioned primers to obtain an amplification fragment containing the ywjH gene 3′ end region and about 1370 bp of the downstream region.
(iii) Amplification of cat Gene by PCR
50-mer PCR primers having the following nucleotide sequences were prepared based on the information from the public gene data bank (GenBank Accession Nos. V01277 and NC—000964).
gagaagcgaatgaattaggaattctAAAGCACCCATTAGTTCAACAAACG (SEQ ID NO: 29, the nucleotides indicated with small letters correspond to the sequence of 5′ end region of the ywjH gene, and they were designed so as to be complementary to the 3′ end region of SEQ ID NO: 26)
ctgtttaacttctgaaatcaggtcaaAGAAGGATATGGATCTGGAGCTGT (SEQ ID NO: 30, the nucleotides indicated with small letters correspond to the sequence of 3′ end region of the ywjH gene, and they were designed so as to be complementary to the 3′ end region of SEQ ID NO: 27)
PCR (98° C. for 10 second, 55° C. for 30 seconds, 72° C. for 1.5 minutes, 30 cycles, Gene Amp PCR System Model 9600, Perkin-Elmer) was performed using the plasmid pC194 carrying the chloramphenicol resistance gene (cat) as the template and the aforementioned primers to obtain an amplification fragment of about 980 bp containing the cat gene.
(iv) Amplification of the Fragment Containing the ywjH Region Which has been Inserted with the cat Gene by Recombinant PCR
The DNA fragments amplified in (i) to (iii) mentioned above were purified using MicroSpin Column S-400 (Amersham Pharmacia Biotech), and then a mixture of these primers in appropriate amounts was used as the template together with the sequences of SEQ ID NOS: 25 and 28 to perform PCR (98° C. for 10 second, 55° C. for 30 seconds, 72° C. for 4 minutes, 30 cycles, Gene Amp PCR System Model 9600, Perkin-Elmer), resulting in a fragment containing the ywjH region inserted with the cat gene.
(v) Preparation of ywjH-Disrupted Inosine-Producing Strain
The DNA fragment containing the ywjH region into which the cat gene (ywjH::cat) obtained in (iv) had been inserted was subjected to agarose gel electrophoresis, and the target fragment was extracted from the gel. The DNA fragment purified as described above was used to transform competent cells of the B. subtilis KMBS321 strain prepared by the method of Dubnau and Davidoff-Abelson (J. Mol. Biol., 1971, 56, 209-221), and colonies grew on an LB agar plate containing 2.5 μg/ml of chloramphenicol and 20 μg/ml of guanine. Chromosomal DNAs were prepared from these colonies, strains in which ywjH region on the chromosome was replaced with the ywjH region containing the chloramphenicol resistance gene (ywjH::cat) by double recombination were identified by the PCR method described in (iv), and one of these strains was designated TABS135.
(3) Construction a Strain Deficient in Both fbp and ywjH
A strain deficient in the transaldolase gene (ywjH) was constructed from the recombinant strain TABS133 as described below. The TABS133 strain, which is derived from Bacillus subtilis (B. subtilis 168 Marburg strain, ATCC 6051), is deficient in the purine operon repressor gene (purR), succinyl-AMP synthase gene (purA), purine nucleoside phosphorylase gene (pupG) and fructose bisphosphatase gene (fbp), and has an attenuated IMP dehydrogenase gene (guaB). The strain also has a modified purine operon promoter region and SD sequence of the PRPP synthetase gene (prs).
(i) Amplification of ywjH Upstream Region by PCR
26-mer and 50-mer PCR primers having the following nucleotide sequences were prepared based on the information from the public gene data bank (GenBank Accession Nos. NC—000964 and M29725).
TAAAGCGTGATAGACATACAGTGCTG (SEQ ID NO: 31) cattgcaagacttttttcaccaagcAGAATTCCTAATTCATTCGCTTCTC (SEQ ID NO: 32, the nucleotides indicated with small letters correspond to the promoter upstream region of the tetracycline resistance gene (tet) cloned in pDG1513)
PCR (98° C. for 10 second, 55° C. for 30 seconds, 72° C. for 2.5 minutes, 30 cycles, Gene Amp PCR System Model 9600, Perkin-Elmer) was performed using the chromosomal DNA of the B. subtilis 168 Marburg strain as the template and the aforementioned primers to obtain an amplification fragment containing the ywjH gene 5′ end region and about 2250 bp of the upstream region.
(ii) Amplification of ywjH Downstream Region by PCR
50-mer and 26-mer PCR primers having the following nucleotide sequences were prepared based on the information from the public gene data bank (GenBank Accession Nos. NC—000964 and M29725).
gagagagttcaaaattgatcctttTTGACCTGATTTCAGAAGTTAAACAG (SEQ ID NO: 33, the nucleotides indicated with small letters correspond to a downstream region of structural gene of the tetracycline resistance gene (tet) cloned in pDG1513)
TTGCATATACATGTCAGGAGCATTCA (SEQ ID NO: 34)
PCR (98° C. for 10 second, 55° C. for 30 seconds, 72° C. for 2.5 minutes, 30 cycles, Gene Amp PCR System Model 9600, Perkin-Elmer) was performed using the chromosomal DNA of the B. subtilis 168 Marburg strain as the template and the aforementioned primers to obtain an amplification fragment containing the ywjH gene 3′ end region and about 2280 bp of the downstream region.
(iii) Amplification of tet Gene by PCR
50-mer PCR primers having the following nucleotide sequences were prepared based on the information from the public gene data bank (GenBank Accession Nos. M29725 and NC—000964).
gagaagcgaatgaattaggaattctGCTTGGTGAAAAAAGTCTTGCAATG (SEQ ID NO: 35, the nucleotides indicated with small letters correspond to the sequence of 5′ end region of the ywjH gene, and they were designed so as to be complementary to the 3′ end region of SEQ ID NO: 32)
ctgtttaacttctgaaatcaggtcaaAAAGGATCAATTTTGAACTCTCTC (SEQ ID NO: 36, the nucleotides indicated with small letters correspond to the sequence of 3′ end region of the ywjH gene, and they were designed so as to be complementary to the 3′ end region of SEQ ID NO: 33)
PCR (98° C. for 10 second, 55° C. for 30 seconds, 72° C. for 2.5 minutes, 30 cycles, Gene Amp PCR System Model 9600, Perkin-Elmer) was performed using the plasmid pDG1513 carrying the tetracycline resistance gene (tet) as a template and the aforementioned primers to obtain an amplification fragment of about 2070 bp containing the tet gene.
(iv) Amplification of the Fragment Containing the ywjH Region Which Contains the tet Gene by Recombinant PCR
The DNA fragments amplified in (i) to (iii) mentioned above were purified by using MicroSpin Column S-400 (Amersham Pharmacia Biotech), and then a mixture of these primers in appropriate amounts was used as the template together with the sequences of SEQ ID NOS: 31 and 34 to perform PCR (98° C. for 10 second, 55° C. for 30 seconds, 72° C. for 7 minutes, 30 cycles, Gene Amp PCR System Model 9600, Perkin-Elmer) and thereby obtain a fragment containing the ywjH gene region containing the tet gene.
(v) Preparation of fbp and ywjH-Disrupted Inosine-Producing Strain
The DNA fragment containing the ywjH region into which the tet gene (ywjH::tet) obtained in (iv) had been inserted was subjected to agarose gel electrophoresis, and the target fragment was extracted from the gel. The DNA fragment purified as described above was used to transform competent cells of the B. subtilis TABS133 strain prepared by the method of Dubnau and Davidoff-Abelson (J. Mol. Biol., 1971, 56, 209-221), and colonies grew on an LB agar plate containing 12.5 μg/ml of tetracycline and 20 μg/ml of guanine. Chromosomal DNAs were prepared from these colonies, and strains in which the ywjH region on the chromosome was replaced with the ywjH region having the tetracycline resistance gene (ywjH::tet) by double recombination were identified by the PCR method described in (iv), and one of these strains was designated TABS174.
(4) Production of a Purine Nucleoside by the Inosine-Producing Strain Deficient in Either the fbp Gene or the ywjH Gene, or Both
The fbp gene-deficient strain TABS133, the ywjH gene-deficient strain TABS135, the strain TABS174 deficient in both genes, and a control strain KMBS321 were each uniformly applied on an LB medium plate containing 20 mg/L of guanine, and cultured overnight at 34° C. The cells on 1⁄8 of the plate were inoculated into 20 ml of fermentation medium in a 500-ml volume Sakaguchi flask, then 50 g/L of calcium carbonate was added to the medium, and the cells were cultured at 34° C. with shaking. Seventy two hours after the start of the culture, the medium was sampled, and amounts of inosine and hypoxanthine present in the medium were measured by known methods. The amount of inosine which had accumulated with the fbp gene-deficient strain TABS133 and the ywjH gene-deficient strain TABS135 were higher than that observed with the KMBS321 control strain. Furthermore, the amount of inosine which had accumulated with the strain deficient in both genes was higher than that observed with the either the TABS133 strain or TABS135 strain.










Composition of fermentation medium



















Glucose
30
g/L



KH2PO4
1
g/L



NH4Cl
32
g/L



Mameno (T-N)*
1.35
g/L



DL-Methionine
0.4
g/L



L-Tryptophan
0.02
g/L



Adenine
0.1 
g/L



Guanosine
0.075
g/L



MgSO4
0.4 
g/L



FeSO4
0.01 
g/L



MnSO4
0.01
g/L



Adekanol (antifoam)
0.5
ml/L



(adjusted to pH 7.0 with KOH)





Calcium Carbonate
50 
g/L







*Soybean protein hydrolysate
















TABLE 1







B. subtilis strains
Inosine (g/L)









KMBS321
5.3



TABS133
5.8



TABS135
6.0



TABS174
6.5










Explanation of Sequence Listing:
SEQ ID NO: 1: Nucleotide sequence of ywjH gene
SEQ ID NO: 2: Amino acid sequence of transaldolase
SEQ ID NO: 3: Nucleotide sequence of prs gene
SEQ ID NO: 4: Amino acid sequence of phosphoribosyl pyrophosphate synthetase
SEQ ID NO: 5: Nucleotide sequence of purR gene
SEQ ID NO: 6: Amino acid sequence of purine repressor
SEQ ID NO: 7: Nucleotide sequence of deoD gene
SEQ ID NO: 8: Amino acid sequence of deoD gene product (purine nucleoside phosphorylase)
SEQ ID NO: 9: Nucleotide sequence of pupG gene
SEQ ID NO: 10: Amino acid sequence of pupG gene product (purine nucleoside phosphorylase)
SEQ ID NO: 11: Nucleotide sequence of purA gene
SEQ ID NO: 12: Amino acid sequence of succinyl-AMP synthase
SEQ ID NO: 13: Nucleotide sequence of guaB gene
SEQ ID NO: 14: Amino acid sequence of IMP dehydrogenase
SEQ ID NO: 15: Nucleotide sequence of fbp gene
SEQ ID NO: 16: Amino acid sequence of fructose bisphosphatase
SEQ ID NO: 17: Primer for purR gene amplification
SEQ ID NO: 18: Primer for purR gene amplification
SEQ ID NO: 19: Primer for fbp gene upstream region amplification
SEQ ID NO: 20: Primer for fbp gene upstream region amplification
SEQ ID NO: 21: Primer for fbp gene downstream region amplification
SEQ ID NO: 22: Primer for fbp gene downstream region amplification
SEQ ID NO: 23: Primer for cat gene amplification
SEQ ID NO: 24: Primer for cat gene amplification
SEQ ID NO: 25: Primer for ywjH gene upstream region amplification
SEQ ID NO: 26: Primer for ywjH gene upstream region amplification
SEQ ID NO: 27: Primer for ywjH gene downstream region amplification
SEQ ID NO: 28: Primer for ywjH gene downstream region amplification
SEQ ID NO: 29: Primer for cat gene amplification
SEQ ID NO: 30: Primer for cat gene amplification
SEQ ID NO: 31: Primer for ywjH gene upstream region amplification
SEQ ID NO: 32: Primer for ywjH gene upstream region amplification
SEQ ID NO: 33: Primer for ywjH gene downstream region amplification
SEQ ID NO: 34: Primer for ywjH gene downstream region amplification
SEQ ID NO: 35: Primer for tet gene amplification
SEQ ID NO: 36: Primer for tet gene amplification
INDUSTRIAL APPLICABILITY
By using the Bacillus bacterium of the present invention, production efficiency of a purine nucleoside and/or a purine nucleotide can be improved.
While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. Each of the aforementioned documents is incorporated by reference herein in its entirety.

## Claims

1. A method for producing a purine-derived substance, which comprises
(A) culturing a bacterium belonging to the genus Bacillus which is able to produce a purine-derived substance in a medium, and
(B) collecting the purine-derived substance from the bacterium or medium, wherein the bacterium has been modified to decrease the enzymatic activity of transaldolase by disrupting a gene encoding transaldolase, or decreasing the expression of the gene encoding transaldolase,
wherein the gene transaldolase is selected from the group consisting of:
(A) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 2, and
(B) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 2, but which includes substitutions, deletions, insertions, or additions of one to 10 amino acid residues and has transaldolase activity,
wherein said bacterium has been further modified to decrease fructose bisphosphatase activity as compared to an unmodified strain by disrupting a gene encoding fructose bisphosphatase, or decreasing the expression of the gene encoding fructose bisphosphatase.

2. The method according to claim 1, wherein the purine-derived substance is a purine nucleoside or a purine nucleotide.

3. The method according to claim 2, wherein the purine-derived substance is a purine nucleoside selected from the group consisting of inosine, xanthosine, guanosine, and adenosine.

4. The method according to claim 2, wherein the purine-derived substance is a purine nucleotide selected from the group consisting of inosinic acid, xanthylic acid, guanylic acid, and adenylic acid.

5. A method for producing a purine nucleotide, which comprises
(A) producing a purine nucleoside by the method according to claim 3,
(B) reacting the purine nucleoside with a phosphate donor selected from the group consisting of polyphosphoric acid, phenyl phosphate, and carbamyl phosphate, and a microorganism which is able to produce a nucleoside-5′-phosphoric acid ester or acid phosphatase to produce a purine nucleotide, and
(C) collecting the purine nucleotide.

6. The method according to claim 1, wherein said bacterium has been further modified to increase phosphoribosyl pyrophosphate synthetase activity by overexpressing a gene encoding a phosphoribosyl pyrophosphate synthetase.

7. The method according to claim 1, wherein said bacterium has been further modified to increase expression of the purine operon by a method selected from the group consisting of overexpressing the purine operon and disrupting the purR gene, wherein said purR gene encodes a repressor of the purine operon.

8. The method according to claim 7, wherein the expression of the purine operon is increased by disrupting the purR gene, wherein said purR gene encodes a repressor of the purine operon.

9. The method according to claim 1, wherein said bacterium has been further modified to decrease purine nucleoside phosphorylase activity by disrupting the gene encoding purine nucleoside phosphorylase, or decreasing the expression of the gene encoding purine nucleoside phosphorylase.

10. The method according to claim 1, wherein said bacterium has been further modified to decrease IMP dehydrogenase activity by disrupting the gene encoding IMP dehydrogenase, or decreasing the expression of the gene encoding IMP dehydrogenase.

11. The method according to claim 1, wherein said bacterium is Bacillus subtilis.