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  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
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  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
  • C12P19/28—N-glycosides
  • C12P19/30—Nucleotides
  • C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
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  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
  • C12P19/28—N-glycosides
  • C12P19/30—Nucleotides
  • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Definitions

• the present invention relates to the microbiological industry, and specifically to a method for producing purine ribonucleosides, which are important as raw materials in the synthesis of purine nucleotides.
• the method uses a Bacillus bacterium which has been modified to attenuate expression of the gene encoding 3-hexulose-6-phosphate synthase.
• mutant strains is conventionally accomplished by subjecting microorganisms to mutagenesis by UV irradiation or by treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), and selecting a strain with the desired characteristics by using a suitable selection medium.
• mutant strains have been obtained by breeding using genetic engineering techniques, for example, for strains of the genera Bacillus (Japanese Patent Application Laid-Open Nos.
• the ribulose monophosphate (RuMP) pathway is one of the metabolic pathways for the synthesis of compounds containing carbon-carbon bonds from one- carbon units, and is found in many methane- and methanol-utilizing bacteria, which are known as methylotrophs.
• the characteristic enzymes of this pathway are 3-hexulose-6- phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), neither of which has been thought to exist outside methylotrophs.
• HPS 3-hexulose-6- phosphate synthase
• PHI 6-phospho-3-hexuloisomerase
• YckG presumed yckG gene product of Bacillus subtilis has a primary structure similar to that of the methylotroph HPS.
• An aspect of the present invention is to improve microorganisms suitable for the production of purine nucleosides by fermentation and to provide a method for producing purine nucleosides using the strains of the microorganism.
• the present invention provides a bacterium belonging to the genus Bacillus, particularly Bacillus subtilis and Bacillus amyloliquefaciens, having an increased ability to produce purine nucleosides.
• HPS hexulose-6-phosphate synthase
• purine nucleoside is selected from the group consisting of inosine, xanthosine, guanosine, and adenosine.
• purine nucleoside is selected from the group consisting of inosine, xanthosine, guanosine, and adenosine.
• purine nucleotide is selected from the group consisting of 5'-inosinic acid, xanthosine-5' -phosphate, 5'-guanylic acid, and 5'-adenylic acid.
• Figure 1 shows the structure and unique restriction sites of the pKSl plasmid.
• the bacterium is able to produce a purine nucleoside, and has been modified to decrease 3-hexulose-6-phosphate synthase activity.
• the bacterium belongs to the genus Bacillus.
• Bacillus bacterium examples include Bacillus subtilis subsp. subtilis strain 168 (B. subtilis 168) or Bacillus amyloliquefaciens (B. amyloliquefaciens).
• B. amyloliquefaciens is a heterogenic species.
• a number of B. amyloliquefaciens strains are known, including SB, T, P, W, F, N, K and H (Welker N.E., Campbell L.L., Unrelatedness of Bacillus amyloliquefaciens and Bacillus subtilis. J. Bacteriol., 94:1124-1130, 1967). Recently, Bacillus strains were isolated from plants, which are considered to be a distinct ecotype of B.
• amyloliquefaciens (Reva et al., Taxonomic characterization and plant colonizing abilities of some bacteria related to Bacillus amyloliquefaciens and Bacillus subtilis. FEMS Microbiol. Ecol., 48:249-259, 2004).
• bacteria belonging to genus Bacillus also include the following: Bacillus licheniformis, Bacillus pumilis, Bacillus megaterium, Bacillus brevis, Bacillus polymixa, and Bacillus stearothermophilus.
• Bacillus licheniformis Bacillus pumilis
• Bacillus megaterium Bacillus brevis
• Bacillus polymixa Bacillus polymixa
• Bacillus stearothermophilus examples of bacteria belonging to genus Bacillus
• the bacteria may also have various nutritional requirements, drug resistances, drug sensitivities, and drug dependencies.
• purine nucleoside includes inosine, xanthosine, guanosine, and adenosine, preferably inosine.
• the phrase "ability to produce a purine nucleoside” means an ability to produce and cause accumulation of a purine nucleoside in a medium.
• the phrase "bacterium has the ability to produce a purine nucleoside” means that the bacterium belonging to the genus Bacillus is able to produce and cause accumulation of purines, such as purine nucleosides in a medium in an amount larger than a wild-type or non-modified strain of a Bacillus bacterium, for example, B. subtilis, such as B. subtilis 168.
• this phrase means that the microorganism is able to produce and cause accumulation in a medium in an amount of not less than 10 mg/1, more preferably not less than 50 mg/1 of purine nucleoside, such as inosine, xanthosine, guanosine, or/and adenosine.
• purine nucleoside such as inosine, xanthosine, guanosine, or/and adenosine.
• Term "activity of HPS” means an activity to catalyze the reaction of the formation D-arabino-3-hexulose-6-phosphate by the Mg 2+ -depending aldol condensation of formaldehyde with ribulose-5-phosphate.
• the HPS activity can be measured as described in Yasueda, H. et al., (J. Bacteriology, 181(23), p. 7154-7160 (1999)).
• the gene coding for HPS from Bacillus subtilis has been elucidated (nucleotides complementary to nucleotides 374725 to 375357 in the sequence of GenBank Accession NC 000964).
• the hxlA gene from B. subtilis is located on the chromosome between the hxlB and hxlR genes.
• the nucleotide sequence of the B. subtilis hxlA gene and the amino acid sequence encoded by the hxlA gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
• the gene coding for HPS from Bacillus amyloliquefaciens has been elucidated (nucleotides complementary to nucleotides 340797 to 341432 in the sequence of GenBank Accession NC 009725).
• the hxlA gene from B. amyloliquefaciens is located on the chromosome between the hxlB and hxlR genes.
• the nucleotide sequence of the B. amyloliquefaciens hxlA gene and the amino acid sequence encoded by the hxlA gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
• the gene can be cloned into a vector that is able to function in a Bacillus bacterium prior to introduction into the bacterium.
• shuttle vectors such as pHY300PLK, pMWMXl, pLF22, or pKSl can be used.
• the wild-type gene is replaced with the mutant by homologous recombination.
• the hxlA gene to be inactivated on the chromosome is not limited to the genes shown in SEQ ID No: 1 and SEQ ID No: 3, but may include genes homologous to SEQ ID No: 1 and SEQ ID No: 3 which encode variant HPS proteins.
• variant HPS protein as used in the present invention means a protein which has changes in the sequence, whether they are deletions, insertions, additions, or substitutions of amino acids, but still maintains the activity of the HPS protein. The number of changes in the variant protein depends on the position in the three dimensional structure of the protein or the type of amino acid residues.
• SEQ ID NO: 2 may be 1 to 30, preferably 1 to 15, and more preferably 1 to 5 in SEQ ID NO: 2 and SEQ ID No:4. These changes can occur in regions of the protein which are not critical for the function of the protein. This is because some amino acids have high homology to one another so the three dimensional structure or activity is not affected by such a change.
• the protein variant encoded by the hxlA gene may be one which has a homology of not less than 80%, preferably not less than 90%, more preferably not less than 95%, still more preferably not less than 98%, and most preferably not less than 99%, with respect to the entire amino acid sequence shown in SEQ ID NO: 2 and SEQ ID NO: 4, as long as the activity of the HPS protein prior to inactivation of the hxlA gene is maintained.
• Homology between two amino acid sequences can be determined using well-known methods, for example, the computer program BLAST 2.0, which calculates three parameters: score, identity and similarity.
• the hxlA gene may be a variant which hybridizes under stringent conditions with the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 3, or a probe which can be prepared from the nucleotide sequence under stringent conditions, provided that it encodes a functional HPS protein prior to inactivation.
• Stringent conditions include those under which a specific hybrid, for example, a hybrid having homology of not less than 60%, preferably not less than 70%, more preferably not less than 80%, more preferably not less than 90%, more preferably not less than 95%, still more preferably not less than 98%, and most preferably not less than 99% is formed and a non-specific hybrid, for example, a hybrid having homology lower than the above, is not formed.
• stringent conditions are exemplified by washing one time or more, preferably two or three times at a salt concentration of IxSSC, 0.1% SDS, preferably 0. IxSSC, 0.1% SDS at 60 0 C.
• Duration of washing depends on the type of membrane used for blotting and, as a rule, can be what is recommended by the manufacturer. For example, the recommended duration of washing for the HybondTM N+ nylon membrane ( Amersham) under stringent conditions is 15 minutes. Preferably, washing may be performed 2 to 3 times.
• the length of the probe may be suitably selected depending on the hybridization conditions, and is usually 100 bp to 1 kbp.
• Expression of the hxlA gene can be attenuated by introducing a mutation into the gene on the chromosome so that intracellular activity of the protein encoded by the gene is decreased as compared with an unmodified strain.
• a mutation can be the replacement of one base or more, resulting in an amino acid substitution in the protein encoded by the gene (missense mutation), introduction of a stop codon (nonsense mutation), deletion of one or two bases to cause a frame shift, insertion of a drug- resistance gene, or deletion of a part of the gene or the entire gene.
• Expression of the hxlA gene can also be attenuated by modifying an expression regulating sequence, such as the promoter, the Shine-Dalgarno (SD) sequence, etc..
• the following methods may be employed to introduce a mutation by gene recombination.
• a DNA fragment containing a mutant gene is prepared and transformed into a bacterium. Then the native gene on the bacterial chromosome is replaced with the mutant gene by homologous recombination, and the resulting strain is selected.
• Such gene replacement using homologous recombination can be conducted by the method employing a linear DNA, which is known as "Red-driven integration" (Datsenko, K.A. and Wanner, B.L., Proc. Natl. Acad. Sci. USA, 97, 12, p 6640-6645 (2000)), or by methods employing a plasmid containing a temperature-sensitive replication origin (U.S.
• Patent 6,303,383 or JP 05-007491 A Furthermore, the incorporation of a site-specific mutation by gene substitution using homologous recombination such as set forth above can also be conducted by transformation with a plasmid lacking the ability to replicate in the host.
• Expression of the gene can also be attenuated by insertion of a transposon or an IS factor into the coding region of the gene (U.S. Patent No. 5,175,107), or by conventional methods, such as by mutagenesis using UV irradiation or treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine).
• Inactivation of the gene can also be performed by conventional methods, such as by mutagenesis using UV irradiation or treatment with nitrosoguanidine (N-methyl- N'-nitro-N-nitrosoguanidine), site-directed mutagenesis, gene disruption using homologous recombination, or/and insertion-deletion mutagenesis.
• mutagenesis using UV irradiation or treatment with nitrosoguanidine (N-methyl- N'-nitro-N-nitrosoguanidine), site-directed mutagenesis, gene disruption using homologous recombination, or/and insertion-deletion mutagenesis.
• the bacterium can be obtained by attenuating the expression of the gene encoding HPS in a bacterium inherently having the ability to produce purine nucleoside.
• the bacterium can be obtained by imparting the ability to produce a purine nucleoside to a bacterium in which the expression of the HPS gene is attenuated.
• Bacillus strain that may be used to derive the Bacillus bacterium is the B. subtilis inosine-producing strain KMBS375.
• KMBS375 Ppur*- ⁇ att ⁇ purA ⁇ purR ⁇ pupG ⁇ deoD guaB24; Reference Example).
• Other parent Bacillus strains include B. subtilis strain AJ 12707 (FERM P- 12951) (Japanese Patent Application JP6113876A2), B.
• subtilis strain AJ3772 (FERM P-2555) (Japanese Patent Application JP62014794A2), Bacillus pumilus NA-1102 (FERM BP-289), Bacillus subtilis NA-6011 (FERM BP-291), Bacillus subtilis Gl 136A (ATCC No. 19222) (US patent 3,575,809), re-identified as Bacillus amyloliquefaciens AJ 1991, deposited on October 3, 2005 at VKPM as Bacillus amyloliquefaciens Gl 136A (VKPM B-8994) and converted into an international deposit on October 13, 2006, Bacillus subtilis NA-6012 (FERM BP-292) (US patent 4,701,413), B.
• B. subtilis strain KMBS 16 may be used. This strain is a derivative of the known B. subtilis 168 trpC2 strain, in which the purR gene coding for a purine repressor (purR: :spc), pur A gene coding for succinyl- AMP synthase (purA::erm), and deoD gene coding for purine nucleoside phosphorylase (deoD::kan) (Russian Patent application No.
• the bacterium may be further improved by enhancing the expression of one or more genes involved in the purine biosynthesis, which include the pur operon of B. subtilis (Ebbole D.J. and Zalkin H. J. Biol. Chem., 262: 8274-87 (1987), Bacillus subtilis and Its Closest Relatives, Editor in Chief: A.L. Sonenshein, ASM Press, Washington D.C., 2002).
• the inosine-producing B. subtilis strain having a modified /?Mr-operon regulatory region has been described (US Patent 7,326,546, 2008).
• Methods for the preparation of chromosomal DNA, hybridization, PCR, preparation of plasmid DNA, digestion and ligation of DNA, transformation, selection of an oligonucleotide as a primer, and the like may be ordinary methods well-known to one skilled in the art. These methods are described in Sambrook, J., and Russell D., "Molecular Cloning A Laboratory Manual, Third Edition", Cold Spring Harbor Laboratory Press (2001), and the like.
• the method for producing a nucleoside includes the steps of cultivating the bacterium in a culture medium, to allow the nucleoside to be produced and accumulated in the culture medium, and collecting the nucleoside from the culture medium.
• the cultivation, collection, and purification of purine nucleosides from the medium and the like may be performed in a manner similar to conventional fermentation wherein a purine nucleoside is produced using a microorganism.
• the culture medium for purine nucleoside production may be a typical medium which contains a carbon source, a nitrogen source, inorganic ions, and other organic components as required.
• the carbon source saccharides such as glucose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose, and hydrolyzates of starches; alcohols such as glycerol, mannitol and sorbitol; organic acids such as gluconic acid, fumaric acid, citric acid and succinic acid and the like can be used.
• inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate
• organic nitrogen such as of soy bean hydrolyzates
• ammonia gas aqueous ammonia, and the like
• vitamins such as vitamin B 1 required substances, for example, organic nutrients such as nucleic acids such as adenine and RNA, or yeast extract, and the like may be present in appropriate, or even trace, amounts.
• organic nutrients such as nucleic acids such as adenine and RNA, or yeast extract, and the like may be present in appropriate, or even trace, amounts.
• small amounts of calcium phosphate, magnesium sulfate, iron ions, manganese ions, and the like may be added, if necessary.
• Cultivation is preferably performed under aerobic conditions for 16 to 72 hours, and the culture temperature during cultivation is controlled to within 30 to 45°C, and the pH within 5 to 8.
• the pH can be adjusted by using an inorganic or organic acidic or alkaline substance, as well as ammonia gas.
• solids such as cells can be removed from the liquid medium by centrifugation or membrane filtration, and then the target purine nucleoside can be recovered from the fermentation liquor by any combination of conventional techniques, such as ion exchange resin and precipitation.
• the method producing purine nucleotides includes the steps of cultivating the bacterium in a culture medium, phosphorylizing the desired purine nucleoside, allowing excretion into the culture medium by the bacterium, and collecting the purine nucleotide. Furthermore, the method for producing 5'-inosinic acid includes the steps of cultivating the bacterium in a culture medium, phosphorylizing inosine, allowing excretion into the culture by the bacterium, and collecting 5'-inosinic acid.
• the method for producing 5'-xanthylic acid includes the steps of cultivating the bacterium in a culture medium, phosphorylizing xanthosine, allowing excretion into the culture medium by the bacterium, and collecting 5'-xanthylic acid.
• the method for producing 5'-guanylic acid includes the steps of cultivating the bacterium in a culture medium, phosphorylizing guanosine, allowing excretion into the culture medium by the bacterium, and collecting 5'-guanylic acid.
• the method for producing 5'-guanylic acid includes the steps of cultivating the bacterium in a culture medium, phosphorylizing xanthosine, allowing excretion into the culture medium by the bacterium, animating 5'-xanthylic acid, and collecting 5'-guanylic acid.
• Purine nucleoside which is to be phosphorylized may be collected from the medium in which the bacterium excreted the purine nucleoside into the medium.
• the medium containing the purine nucleoside can be used for phosphorilization reaction without isolation or purification of the purine nucleoside.
• the cultivation, the collection and purification of inosine from the medium and the like may be performed in a manner similar to conventional fermentation methods wherein inosine is produced using a microorganism.
• the steps of phosphorylating inosine, allowing excretion into the culture medium by the bacterium, and collecting 5'-inosinic acid may be performed in a manner similar to conventional fermentation methods, wherein a purine nucleotide such as 5'-inosinic acid is produced from a purine nucleoside such as inosine.
• the phosphorylation of the purine nucleoside can be performed enzymatically using different phosphatases, nucleoside kinases, or nucleoside phosphotransferases, or chemically using phosphorylating agents such as POCl 3 , or the like.
• a phosphatase which is able to catalyze the C-5' -position selective transfer of a phosphoryl group of pyrophosphate to nucleosides (Mihara et. al, Phosphorylation of nucleosides by the mutated acid phosphatase from Morganella morganii. Appl. Environ.
• phosphatase which is able to catalyze the transfer of a phosphoryl group to the C-2', 3', or 5 '-position of nucleosides utilizing p-nitrophenyl phosphate (Mitsugi, K., et al, Agric. Biol.
• nucleoside kinase guanosine/inosine kinase from E. coli (Mori, H. et. al. Cloning of a guanosine-inosine kinase gene of Escherichia coli and characterization of the purified gene product. J. Bacteriol.
• nucleoside phosphotransferases described by Hammer- Jespersen, K. (Nucleoside catabolism, p. 203-258. In A Munch-Petesen (ed.), Metabolism of nucleotides, nucleosides, and nucleobases in microorganism. 1980, Academic Press, New York) or the like may be used.
• the chemical phosphorylation of nucleosides may be performed using a phosphorylation agent such as POCl 3 (Yoshikawa, K. et. al. Studies of phosphorylation. III. Selective phosphorylation of unprotected nucleosides. Bull.
• the animation of 5'-xanthylic acid can be performed enzymatically using, for example, GMP synthetase from E. coli (Fujio et. al. High level of expression of XMP aminase in Escherichia coli and its application for the industrial production of 5'- guanylic acid. Biosci. Biotech. Biochem. 1997, 61 :840-845; ⁇ P0251489B1).
• Example 1 Construction of a strain with an inactivated hxlA gene
• Primers Pl and P2 contain recognition sites for endonucleases Sacll and Pstl, respectively. Amplification of a 923 bp fragment in the downstream region of the hxlA gene was performed by PCR using primers P3 (SEQ ID NO: 7) and P4 (SEQ ID NO: 8), and the chromosomal DNA of B. subtilis KMBS375 as the template. Primers P3 and P4 contain recognition sites for endonucleases CIaI and Kpnl, respectively. The purified PCR product using Pl and P2 was digested with endonucleases Sacll and Pstl, and then cloned into the corresponding sites of the pKSl plasmid.
• the purified PCR product using P3 and P4 was digested with endonucleases Clal and Kpnl, and also cloned into the corresponding sites of the pKSl plasmid. After cloning both fragments, the plasmid containing the Km R cassette and the upstream and downstream regions of the hxlA gene was digested with endonucleases Sacll and Kpnl and the resulting fragment (3194 bp) was subcloned into the nonreplicative pBluescript II KS vector (Stratagene) from Bacillus. The resulting plasmid was used to transform B. subtilis KMBS375, and 12 Km R transformants were isolated.
• the B. subtilis strains KMBS375 and KMBS375 ⁇ hxl::Km were each cultivated at 34°C for 18 hours in L-broth, and then 0.3 ml of the culture was inoculated into 3 ml of a fermentation medium in a 20 ⁇ 200 mm test tube, and cultivated at 34°C for 72 hours on a rotary shaker. Results are shown in Table 1.
• the amount of inosine which accumulated in the medium was determined by HPLC.
• KMBS375 ⁇ hxl::Km produced a higher amount of inosine (HxR), as compared with KMBS375.
• B. subtilis 168 Marburg (ATCC6051) is a Trp-auxotroph due to the mutant allele trpC2 (trpC; indole-3-glycerol phosphate synthase gene) on the chromosome (Albertini A. M., and A. Galizzi. 1999.
• the B. subtilis 168 Marburg strain can be obtained from the American Type Culture Collection (ATCC) (P.O. Box 1549 Manassas, VA 20108, United States of America).
• the sequence of the trp operon of Bacillus subtilis 168 (trpC2) revisited. Microbiology. 145:3319-3320).
• the trpCl allele with the three nucleotides "art" following position 328 from the trpC2 start codon was PCR-amplified and introduced into B. subtilis 168 Marburg, as follows.
• PCR primers 7 SEQ ID NO. 11
• 8 SEQ ID NO. 12
• PCR (94°C, 30 seconds; 53°C, 1 minute; 72°C, 1 minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was conducted using the above- described primers and a template of the chromosomal DNA of B. subtilis 168 Marburg to amplify fragments containing the 5 '-end region and the upstream region of the trpCf allele.
• PCR primers 9 SEQ ID NO. 13
• 10 SEQ ID NO. 14
• PCR (94°C, 30 seconds; 53°C, 1 minute; 72°C, 1 minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was conducted using the above- described primers and a template of the chromosomal DNA of B. subtilis 168 Marburg to amplify fragments containing the 3 '-end region and the downstream region of the trpC allele.
• the trpC+ allele fragment was extracted from the gel following agarose gel electrophoresis. Competent cells of B. subtilis 168 Marburg prepared as described by Dubnau and Davidoff-Abelson (Dubnau, D., and R. Davidoff-Abelson, J. MoI. Biol., 56:209-221 (1971)) were transformed with the DNA fragment, and colonies that were capable of growing on a minimal medium agar plate were selected. Chromosomal DNA was prepared from these colonies. DNA fragments were amplified by PCR using the chromosomal DNA as the template and primers of 7 (SEQ ID NO. 11) and 10 (SEQ ID NO.
• the recombinant thus obtained was not a Trp-auxotroph, and the strain was named KMBS275.
• PCR primers 11 SEQ ID NO. 15
• 12 SEQ ID NO. 16
• Primer 38 contains the Ec ⁇ RI site at the 5'-end thereof.
• Primer 38 contains a junction created by the in-frame deletion.
• PCR (94 0 C, 30 seconds; 55°C, 1 minute; 72°C, 1 minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was conducted using the above-described primers and a template of the chromosomal DNA of B. subtilis 168 Marburg to amplify fragments containing the 5 '-end region and the upstream region of AhisC.
• Primer 40 contains a junction created by the in-frame deletion.
• Primer 41 contains a BamHl site at the 5 '-end thereof.
• PCR (94°C, 30 seconds; 55 0 C, 1 minute; 72°C, 1 minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was conducted using the above-described primers and a template of the chromosomal DNA of B. subtilis 168 Marburg to amplify fragments containing the 3 '-end region and the downstream region of the AhisC.
• the two DNA fragments amplified as described above were purified using a MicroSpin Column S- 400 (Amersham Pharmacia Biotech). PCR was conducted using a suitable quantity of a mixture of the two fragments as the template, and the primers 11 (SEQ ID NO. 15) and 14 (SEQ ID NO. 18) (94 0 C, 30 seconds; 55 0 C, 1 minute; 72 0 C, 2 minutes; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)). An amplified fragment (about 1.5 kb) of the AhisC was obtained.
• the amplified fragment was digested with Ec ⁇ RI and BamKl (37 0 C, overnight) and separated using an agarose gel.
• the target fragment was extracted from the gel and ligated to the B. subtilis chromosomal integration vector pJPMl (Mueller, J.P., G. Bukusoglu, and A. L. Sonenshein. 1992. Transcriptional regulation of Bacillus subtilis glucose starvation-inducible genes: control of gsiA by the ComP-ComA signal transduction system. J. Bacteriol. 174:4361-4373.), which had been digested with the same enzymes (37 0 C, 3 hrs).
• a plasmid containing the correctly inserted AhisC gene was selected, confirmed to have no other PCR-derived mutations by DNA sequencing, and named pKM186.
• Competent cells of the B. subtilis 168 Marburg strain prepared as described above were transformed with pKM186, and the colonies (single-crossover recombinants) that were capable of growing on an LB agar plate containing 2.5 ⁇ g/mL of chloramphenicol (Cm) were selected.
• One of the single-crossover recombinants was inoculated into 10 mL of LB medium supplemented with 20 mg/L of Guanine (LB+Gua medium) and successively subcultured for 2 days at 37 0 C. Colonies exhibiting chloramphenicol sensitivity were selected by using LB+Gua agar medium with/out Cm. Chromosomal DNA was prepared from the Cm-sensitive colonies. PCR was conducted in the same manner as described above using 11 (SEQ ID NO. 15) and 14 (SEQ ID NO. 18). Strains in which the hisC gene on the chromosome had been replaced with the disrupted-type hisC gene (AhisC) by double-crossover recombination were identified. The double-recombinant strain was named KMBS276 (AhisC trpC2).
• the disrupted-type pupG, pur R, and pur A genes (guanosine/inosine phosphorylase, purine operon repressor, succinyl AMP synthase, accordingly) were successively introduced into the recombinant KMBS276 strain (AhisC trpCI) derived from B. subtilis 168 Marburg, and a prototroph was obtained from this strain, as follows.
• PCR primers 15 SEQ ID NO. 19
• 16 SEQ ID NO. 20
• Primer 42 contains a BamRl site at the 5 '-end thereof.
• Primer 43 contains a junction created by the in-frame deletion.
• PCR (94 0 C, 30 seconds; 55 0 C, 1 minute; 72 0 C, 1.5 minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was conducted using the above- described primers and a template of the chromosomal DNA of B. subtilis 168 Marburg to amplify fragments containing the 5 '-end region and the upstream region of ApupG.
• PCR primers 17 SEQ ID NO. 21 and 18 (SEQ ID NO. 22) were designed based on the information from GenBank (Accession No. NC 000964):
• Primer 44 contains a junction created by the in-frame deletion.
• Primer 45 contains a BamRl site at the 5 '-end thereof.
• PCR (94 0 C, 30 seconds; 5O 0 C, 1 minute; 72°C, 1.5 minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was conducted using the above- described primers and a template of the chromosomal DNA of B. subtilis 168 Marburg to amplify fragments containing the 3 '-end region and the downstream region of the ApupG.
• the two DNA fragments amplified as described above were purified using a MicroSpin Column S- 400 (Amersham Pharmacia Biotech). PCR was conducted using a suitable quantity of a mixture of the two fragments as the template, and the primers 15 (SEQ ID NO. 19) and 18 (SEQ ID NO. 22) (94 0 C, 30 seconds; 55°C, 1 minute; 72°C, 3 minutes; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)). An amplified fragment (about 2 kb) of ApupG was obtained.
• the amplified fragment was digested with BamRl (37 0 C, overnight) and separated using an agarose gel.
• the target fragment was extracted from the gel and ligated to the B. subtilis chromosome integration vector pJPMl (J. Bacterid. 174:4361- 4373), which had been digested with the same enzymes (37°C, 3 hrs), followed by treatment with calf intestine phosphatase.
• pJPMl J. Bacterid. 174:4361- 4373
• a plasmid containing the correctly inserted ApupG gene was selected, confirmed to have no other PCR-derived mutations by DNA sequencing, and named pKM199.
• Competent cells of the KMBS276 strain prepared as described above were transformed with pKM199, and the colonies (single-crossover recombinants) that were capable of growing on an LB agar plate containing 2.5 ⁇ g/mL of Cm were selected.
• One of the single-crossover recombinants was inoculated into 10 mL of LB+Gua medium and successively subcultured for 2 days at 37 0 C. Colonies exhibiting chloramphenicol sensitivity were selected by using LB+Gua agar medium with/out Cm. Chromosomal DNA was prepared from the Cm-sensitive colonies. PCR was conducted in the same manner as described above using primers 15 (SEQ ID NO. 19) and 18 (SEQ ID NO. 22). Strains in which the pupG gene on the chromosome had been replaced with the disrupted-type pupG gene ⁇ ApupG) by double-crossover recombination were identified.
• the double-recombinant strain was named KMBS334 ⁇ ApupG AhisC trpCI).
• PCR primers 19 SEQ ID NO. 23 and 20 (SEQ ID NO. 24) were designed based on the information from GenBank (Accession No. NC 000964).
• Primer 46 contains a BamEI site at the 5 '-end thereof.
• Primer 47 contains a junction created by the in-frame deletion.
• PCR (94°C, 30 seconds; 55°C, 1 minute; 72 0 C, 1.5 minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was conducted using the above- described primers and a template of the chromosomal DNA of B. subtilis 168 Marburg to amplify fragments containing the 5 '-end region and the upstream region of ApurR.
• PCR primers primers 21 SEQ ID NO. 25 and 22 (SEQ ID NO. 26) were designed based on the information from GenBank (Accession No. NC 000964).
• Primer 48 contains a junction created by the in-frame deletion.
• Primer 49 contains a BamHl site at the 5'- end thereof.
• PCR (94 0 C, 30 seconds; 5O 0 C, 1 minute; 72°C, 1.5 minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was conducted using the above- described primers and a template of the chromosomal DNA of B. subtilis 168 Marburg to amplify fragments containing the 3 '-end region and the downstream region of the ApurR .
• the two DNA fragments amplified as described above were purified using a MicroSpin Column S- 400 (Amersham Pharmacia Biotech). PCR was conducted using a suitable quantity of a mixture of the two fragments as the template, and the primers 19 (SEQ ID NO. 23) and 22 (SEQ ID NO. 26) (94 0 C, 30 seconds; 55°C, 1 minute; 72 0 C, 2.5 minutes; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)). An amplified fragment (about 2.1 kb) of ApurR was obtained.
• Competent cells of the KMB S334 strain prepared as described above were transformed with pKM200, and the colonies (single-crossover recombinants) that were capable of growing on an LB agar plate containing 2.5 ⁇ g/mL of Cm were selected.
• One of the single-crossover recombinants was inoculated into 10 mL of LB+Gua medium and successively subcultured for 2 days at 37°C. Colonies exhibiting chloramphenicol sensitivity were selected by using LB+Gua agar medium with/out Cm. Chromosomal DNA was prepared from the Cm-sensitive colonies. PCR was conducted in the same manner as described above using primers 19 (SEQ ID NO. 23) and 22 (SEQ ID NO. 26). Strains in which the purR gene on the chromosome had been replaced with the disrupted-type pur R gene (ApurR) by double-crossover recombination were identified. The double-recombinant strain was named KMBS337 (ApurR ApupG AhisC trpCI).
• PCR primers 23 SEQ ID NO. 27
• 24 SEQ ID NO. 28
• Primer 50 contains a BamHl site at the 5 '-end thereof.
• Primer 51 contains a junction created by the in-frame deletion.
• PCR (94 0 C, 30 seconds; 55 0 C, 1 minute; 72 0 C, 1 minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was conducted using the above-described primers and a template of the chromosomal DNA of B. subtilis 168 Marburg to amplify fragments containing the 5 '-end region and the upstream region of the ApurA.
• PCR primers primers 25 SEQ ID NO. 29 and 26 (SEQ ID NO. 30) were designed based on the information from GenBank (Accession No. NC 000964).
• Primer 52 contains a junction created by the in-frame deletion.
• Primer 53 contains a BamRl site at the 5'- end thereof.
• PCR (94 0 C, 30 seconds; 55 0 C, 1 minute; 72°C, 1 minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was conducted using the above-described primers and a template of the chromosomal DNA of B. subtilis 168 Marburg to amplify fragments containing the 3 '-end region and the downstream region of the ApurA.
• the two DNA fragments amplified as described above were purified using a MicroSpin Column S- 400 (Amersham Pharmacia Biotech). PCR was conducted using a suitable quantity of a mixture of the two fragments as the template, and the primers 23 (SEQ ID NO. 27) and 26 (SEQ ID NO. 30) (94 0 C, 30 seconds; 55 0 C, 1 minute; 72°C, 2 minutes; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)). An amplified fragment (about 1.4 kb) of ApurA was obtained.
• the amplified fragment was digested with BamHl (37 0 C, overnight) and separated using an agarose gel.
• the target fragment was extracted from the gel and ligated to the B. subtilis chromosome integration vector pJPMl (J. Bacteriol. 174:4361- 4373) which had been digested with the same enzymes (37 0 C, 3 hrs), followed by treatment with calf intestine phosphatase.
• pJPMl J. Bacteriol. 174:4361- 4373
• a plasmid containing the correctly inserted ApurA gene was selected, confirmed to have no other PCR-derived mutations by DNA sequencing, and named pKM208.
• Competent cells of the KMBS337 strain prepared as described above were transformed with pKM208, and the colonies (single-crossover recombinants) that were capable of growing on an LB agar plate containing 2.5 ⁇ g/mL of Cm were selected.
• One of the single-crossover recombinants was inoculated into 10 niL of LB+Gua medium and successively subcultured for 2 days at 37°C. Colonies exhibiting chloramphenicol sensitivity were selected by using LB+Gua agar medium with/out Cm.
• Chromosomal DNA was prepared from the Cm-sensitive colonies. PCR was conducted in the same manner as described above using primers 23 (SEQ ID NO. 27) and 26 (SEQ ID NO. 30).
• the double-recombinant strain was named KMBS349 (ApurA ApurR ApupG AhisC trpCI).
• Competent cells of the KMBS349 strain prepared as described above were transformed with chromosomal DNA from KMBS275 (see ⁇ Construction of prototroph of B. subtilis 168 Marburg>), and the colonies that were capable of growing on a minimal medium agar plate containing 20 mg/L of adenine (Ade) were selected.
• Strains with adenine auxotrophy were selected from obtained recombinants using the minimal medium agar plate with/out Ade. Then, by colony PCR (PCR conditions for the corresponding genes, pupG, pur R, and pur A are described above), strains in which the three genes on the chromosome had been replaced with the disrupted-type genes (ApupG, ApurR, and ApurA) were identified. The strain was named KMBS350 (ApurA ApurR ApupG).
• guanine-auxotrophic leaky mutation gu ⁇ B24 (the same as guaB(Al) described in US2006275874A1) in the gu ⁇ B gene coding for IMP dehydrogenase was introduced into the recombinant KMBS349 strain (ApurA ApurR ApupG AhisC trpCI) derived from B. subtilis 168 Marburg, and a prototroph was obtained from the resulting strain, as follows.
• Competent cells of the KMBS349 strain (ApurA ApurR ApupG AhisC trpCI) prepared as described above were transformed with the chromosomal DNA from KMBS 193 (guaBv.kan trpC2; US2006275874A1), and the colonies that were capable of growing on plates of LB medium supplemented with 2.5 ⁇ g/ml of kanamycin (Kan) were selected.
• Kan-resistant strains with Gua auxotrophy were identified from the recombinants.
• the obtained strain was named KMBS351 ⁇ guaBv.kan ApurA ApurR ApupG AhisC trpCT).
• Competent cells of the KMBS351 strain (gu ⁇ Bv.k ⁇ n ApurA ApurR ApupG AhisC trpCI) prepared as described above were transformed with chromosomal DNA of YMBS9 (gu ⁇ B24 trpC2; Competent cells of KMBS 193 were transformed with PCR- amplified DNA of a region containing gu ⁇ B in a derivative strain of B. subtilis 168 Marburg, which contains the gu ⁇ B24 mutation on the chromosome, obtaining YMBS9), and the colonies that were capable of growing on a minimal medium agar plate containing 20 mg/L of Ade, His, and Tip were selected.
• Strains with Ade and His auxotrophy were selected from the recombinants using the minimal medium agar plate with/out Ade+His. Then, by colony PCR (PCR conditions for the corresponding genes pupG and pur R were described above) and DNA sequencing, strains in which the two genes on the chromosome had been replaced with the disrupted-type genes (ApupG and ApurR), and that gu ⁇ Bv.k ⁇ n was correctly replaced with gu ⁇ B24, were identified. The obtained strain was named KMBS353 (gu ⁇ B24 ApurA ApurR ApupG AhisC trpCI).
• Competent cells of the KMBS353 strain prepared as described above were transformed with chromosomal DNA of KMBS350 (see 1. Construction of KMBS350 (ApurA ApurR ApupG)), and the colonies that were capable of growing on a minimal medium agar plate containing 20 mg/L of Ade were selected.
• Competent cells of the KMBS337 strain (ApurR ApupG AhisC trpCI) prepared as described above were transformed with the chromosomal DNA of KMBS 198 (Vpurv.cat trpC2; US2006275874A1), and the colonies that were capable of growing on LB+Gua+Cm medium were selected.
• Strains with Ade and His auxotrophy were selected from the recombinants using the minimal medium agar plate with/out Ade+His. Then, by colony PCR (PCR conditions to corresponding genes, pupG and pur R were described above), strains in which the two genes on the chromosome had been replaced with the disrupted-type genes (ApupG and ApurR) were identified. The obtained strain was named KMBS340 (Ppur.-.cat ApurR ApupG AhisC trpC2).
• Competent cells of the KMBS340 strain (Ppur::cat ApurR ApupG AhisC trpC2) prepared as described above were transformed with the chromosomal DNA from KMBS261 (P/wrl- ⁇ att purRv.spc ApupG* trpC2; US2006275874A1), and the colonies that were capable of growing on a minimal medium agar plate containing 20 mg/L of T ⁇ and His were selected.
• Strains with Ade and His auxotrophy were selected from the recombinants using the minimal medium agar plate with/out Ade+His. Then, by colony PCR (PCR conditions for the corresponding genes pupG and pur R were described above) and DNA sequencing, strains in which the two genes on the chromosome had been replaced with the disrupted-type genes ⁇ ApupG and ApurR), and the Ppur*-Aatt was correctly replaced with Vpurv.c ⁇ t, were identified. The obtained strain was named KMBS352 (P/?wr*- ⁇ att ApurR ApupG AhisC trpCI).
• Competent cells of the KMBS352 strain (P/w*- ⁇ att ApurR ApupG AhisC trpC2) prepared as described above were transformed with chromosomal DNA of KMBS350 ⁇ ApurA ApurR ApupG; see 1. Construction of KMBS350 ⁇ ApurA ApurR ApupG)), and the colonies that were capable of growing on a minimal medium agar plate containing 20 mg/L of His and Ade were selected.
• Competent cells of the KMBS359 strain ⁇ Ppur*-Aatt ⁇ purA ApurR ApupG) prepared as described above were transformed with chromosomal DNA of KMBS351 ⁇ gu ⁇ Bwk ⁇ n ApurA ApurR ApupG AhisC trpC2; see 2.(1) Construction of a gu ⁇ B- disrupted strain from KMBS349), and the colonies that were capable of growing on plates of LB+Gua medium supplemented with 2.5 ug/ml of Kan were selected.
• Kan-resistant strains with Gua auxotrophy were identified from the recombinants. Then, by PCR (PCR conditions for Ppur was described in US2006275874A1), strains in which ?pur*-Aatt was not replaced with the wild-type Ppur were selected. The obtained strain was named KMBS364 ⁇ guaB/.kan Ppwr*- ⁇ att ⁇ purA ApurR ApupG).
• Competent cells of the KMBS364 strain (guaBwkan Ppwr*- ⁇ att ⁇ purA ApurR ApupG) prepared as described above were transformed with chromosomal DNA of KMBS356 (guaB24 ApurA ApurR ApupG; see 2.(3) Introduction o ⁇ trpC+ and hisC+ into KMBS353), and the colonies that were capable of growing on a minimal medium agar plate containing 20 mg/L of Ade were selected.
• strains in which the guaBv.kan allele was correctly replaced with guaB24, and F pur* -AdAX was not replaced with the wild-type Ppur were identified.
• the obtained strain was named KMBS365 (guaB24 Ppur* - ⁇ att ⁇ purA ApurR ApupG).
• PCR primers 27 SEQ ID NO. 31 and 28 (SEQ ID NO. 32) were designed based on the information from GenBank (Accession No. NC 000964).
• Primer 54 contains a BamHl site at the 5 '-end thereof.
• Primer 55 contains a junction created by the in-frame deletion.
• PCR (94 0 C, 30 seconds; 55 0 C, 1 minute; 72°C, 1.5 minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was conducted using the above- described primers and a template of the chromosomal DNA of B. subtilis 168 Marburg to amplify fragments containing the 5 '-end region and the upstream region of AdeoD.
• PCR primers 29 SEQ ID NO. 33
• 30 SEQ ID NO. 34
• Primer 56 contains a junction created by the in-frame deletion.
• Primer 57 contains a BamHl site at the 5 '-end thereof.
• PCR (94 0 C, 30 seconds; 55 0 C, 1 minute; 72°C, 1 minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) was conducted using the above-described primers and a template of the chromosomal DNA of B. subtilis 168 Marburg to amplify fragments containing the 3 '-end region and the downstream region of ⁇ deoD.
• the two DNA fragments amplified as described above were purified using a MicroSpin Column S- 400 (Amersham Pharmacia Biotech). PCR was conducted using a suitable quantity of a mixture of the two fragments as the template, and the primers 27 (SEQ ID NO. 31) and 30 (SEQ ID NO. 34) (94 0 C, 30 seconds; 5O 0 C, 1 minute; 72°C, 3 minutes; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)). An amplified fragment (about 2.0 kb) of AdeoD was obtained.
• the amplified fragment was digested with BarnRl (37 0 C, overnight) and separated using an agarose gel.
• the target fragment was extracted from the gel and ligated to the B. subtilis chromosome integration vector pJPMl (J. Bacteriol. 174:4361- 4373) which had been digested with the same enzymes (37 0 C, 3 hrs), followed by treatment with calf intestine phosphatase.
• pJPMl J. Bacteriol. 174:4361- 4373
• a plasmid containing the correctly inserted AdeoD gene was selected, confirmed to have no other PCR-derived mutations by DNA sequencing, and named pKM201.
• Competent cells of the KMBS365 strain prepared as described above were transformed with pKM201, and the colonies (single-crossover recombinants) that were capable of growing on an LB+Gua agar plate containing 2.5 ⁇ g/mL of Cm were selected.
• One of the single-crossover recombinants was inoculated into 10 mL of LB+Gua medium and successively subcultured for 2 days at 37 0 C. Colonies exhibiting Cm sensitivity were selected by using LB+Gua agar medium with/out Cm. Chromosomal DNA was prepared from the Cm-sensitive colonies. PCR was conducted in the same manner as described above using primers 27 (SEQ ID NO. 31) and 30 (SEQ ID NO. 34). Strains in which the deoD gene on the chromosome had been replaced with the disrupted-type deoD gene (AdeoD) by double-crossover recombination were identified. The double-recombinant strain was named KMBS375 (AdeoD guaB24 P/? «r*- ⁇ att ⁇ purA ApurR ApupG).

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