Method for producing mutan, and sucrose-non-assimilating bacterium introduced with gene encoding glucosyltransferase j

Abstract

To provide a mutan production method that eliminates the need for purification of mutan synthase.SOLUTION: Provided are a method for producing mutan, comprising: (i) introducing a gene encoding glucosyltransferase J (GtfJ) into a sucrose-non-assimilating bacterium to construct a GtfJ-expressing strain; (ii) culturing the expressing strain in the presence of sucrose; and (iii) collecting mutan from the culture; and a Ralstonia eutropha strain into which a gene encoding glucosyltransferase (GtfJ) has been introduced, or a Ralstonia eutropha strain, derived from the Ralstonia eutropha into which the gene has been introduced, in which a gene encoding polyhydroxyalkanoate synthase has been deleted.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a method for producing mutan from sucrose using a sucrose-non-assimilating bacterium, and also to a sucrose-non-assimilating bacterium into which a gene encoding glucosyltransferase J has been introduced, particularly a Ralstonia eutropha strain into which the gene has been introduced. [Background technology]

[0002] Mutan is a water-insoluble polymeric polysaccharide in which glucose units are linked by α-1,3-bonds, and in nature exists as a glucan produced by oral streptococci such as Streptococcus mutans and Streptococcus sobrinus, which are known to cause dental caries.

[0003] In recent years, mutan has been found to be useful in oral compositions that inhibit plaque formation and as a moisturizing agent, and due to its water-insoluble nature, it is attracting attention as an extremely unique material that may also be used as a carrier, etc.

[0004] Conventionally, to obtain mutan, oral streptococci have been cultured in the presence of sucrose to obtain glucan (a mixture of α-1,3-glucan and α-1,6-glucan), and then the α-1,6-glucan has been decomposed and removed using dextranase (Non-patent Document 1). However, not only does it take a long time to process α-1,6-glucan, but there is also the risk of contamination with various bacteria, making it difficult to produce high-quality mutan industrially. To solve this problem, an improved method has been reported in which dextranase is added during the culture process (Patent Document 1).

[0005] Apart from such fermentation methods, it is theoretically possible to isolate and purify only the mutan synthase (glucosyltransferase-I (hereinafter, GTF-I)) from bacterial cells and use it in mutan production; however, in reality, problems such as enzyme aggregation, adsorption, and proteolysis arise. Therefore, instead of the fermentation method using oral streptococci, a method has been reported in which recombinant Escherichia coli carrying a gene encoding GTF-I is cultivated, GTF-I is extracted from the bacterial cells, and GTF-I is added to sucrose (Patent Document 2). Extraction of GTF-I from a recombinant strain differs from isolation and purification from the original bacterial strain in that a large amount of enzyme is obtained and the enzyme does not aggregate. However, the method using the enzyme still requires a lot of time and effort for separation and purification. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent Publication No. 10-262692 [Patent Document 2] Patent Publication No. 2000-83665 [Non-patent literature]

[0007] [Non-Patent Document 1] Dental Caries and Periodontal Disease Vol.2, 1982, Dental Review Company Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, a method for producing mutan that does not require purification of the mutan synthase has been desired. [Means for solving the problem]

[0009] In light of these circumstances, the present inventors conducted extensive research and discovered that glucosyltransferase J (hereinafter referred to as "GtfJ") found in Streptococcus salivarius selectively synthesizes α-1,3-glucan. They then introduced a gene encoding GtfJ into a sucrose-non-utilizing bacterium to create a GtfJ-expressing strain, and found that by culturing the strain in the presence of sucrose, mutan that does not contain α-1,6-glucan can be efficiently obtained, thereby completing the present invention.

[0010] That is, the present invention provides the following. [1] (i) introducing a gene encoding glucosyltransferase J (GtfJ) into a sucrose-non-utilizing bacterium to prepare a GtfJ-expressing strain; (ii) culturing the expression strain in the presence of sucrose; and (iii) collecting mutans from the culture A method for producing mutan, comprising: [2] The method according to [1], wherein the sucrose non-utilizing bacteria are hydrogen-oxidizing bacteria. [3] The method according to [2], wherein the hydrogen-oxidizing bacterium is a Ralstonia eutropha strain or a modified Ralstonia eutropha strain in which a gene encoding polyhydroxyalkanoate (PHA) synthase has been deleted from Ralstonia eutropha. [4] The method described in [3], wherein the modified Ralstonia eutropha strain is used as the hydrogen-oxidizing bacterium, and only mutan is produced. [5] The method according to [3], wherein the hydrogen-oxidizing bacteria is a Ralstonia eutropha strain, and PHA is further produced using fructose produced as a by-product in step (ii) as a carbon source. [6] The method according to any one of [1] to [5], wherein the amount of sucrose added is 0.1 to 20% by weight. [7] A Ralstonia eutropha strain into which a gene encoding glucosyltransferase (GtfJ) has been introduced, or a Ralstonia eutropha strain into which the gene has been introduced and from which the gene encoding polyhydroxyalkanoate synthase has been deleted. [Effects of the Invention]

[0011] The method of the present invention enables efficient production of high-quality mutan. [Brief explanation of the drawings]

[0012] [Figure 1] Figure 1 is a schematic diagram of the pBBREE"32d13dPB_gtfJ vector used to construct the GtfJ-expressing strain. "GtfJ" represents the gene encoding glucosyltransferase J obtained from Streptococcus salivarius ATCC 25975, "lac" represents the lac promoter, "rep" represents the rep gene, "mob" represents the mob gene, "Kmr" represents the kanamycin resistance gene, "NdeI" represents the NdeI restriction enzyme site, and "BamHI" represents the BamHI restriction enzyme site. Figure 1 also shows that the pBBREE"32d13dPB_gtfJ vector was constructed by ligating the pEX-A212_gtfJ vector encoding the GtfJ gene with the pBBREE"32d13dPB_NSDG vector. "Ampr" indicates the ampicillin resistance gene, and "phaCNSDG" indicates the gene encoding the PHA synthase, in which the 149th asparagine of polyhydroxyalkanoate synthase (PhaC) derived from Aeromonas caviae (GenBank accession number: D88825) is replaced with serine, and the 171st aspartic acid of PhaC is replaced with glycine. [Figure 2]Figure 2 shows the growth curves of the Ralstonia eutropha PHB-4 strain under various conditions. -□- indicates the growth curve of R. eutropha PHB-4 / gtfJ (1% sucrose added by weight), -△- indicates the growth curve of the positive control, i.e., R. eutropha PHB-4 / gtfJ (0.5% fructose added by weight) into which the gtfJ gene had been introduced, and -◯- indicates the growth curve of R. eutropha PHB-4 (1% sucrose added by weight) without the gtfJ gene. [Figure 3] Figure 3 (A) shows the growth curve of R. eutropha PHB-4 / gtfJ, and (B) shows the growth curve of R. eutropha H16 / gtfJ, into which the gtfJ gene was introduced. In both figures, -△- indicates the addition of 5% sucrose by weight, and -□- indicates the addition of 10% sucrose by weight. [Figure 4] Figure 4 (A-1), (A-2), and (A-3) show the changes in sucrose (-〇-) and glucose (-□-) or fructose (-△-) concentrations over time in R. eutropha PHB-4 / gtfJ. Sucrose hydrolysis produces glucose and fructose, which are simultaneously consumed by the bacteria, resulting in a "change in concentration." Figures (B-1), (B-2), and (B-3) show the changes in sucrose (-〇-) and glucose (-□-) or fructose (-△-) concentrations over time in R. eutropha H16 / gtfJ. In Figure 4, "amount (g)" indicates the weight of sucrose and other substances in 100 mL of culture medium. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be described in detail below with reference to specific embodiments. However, the present invention is not limited to the following embodiments and can be practiced with appropriate modifications.

[0014] A first embodiment of the present invention relates to a method for producing mutan, (i) introducing a gene encoding glucosyltransferase J (GtfJ) into a sucrose-non-utilizing bacterium to prepare a GtfJ-expressing strain; (ii) culturing the expression strain in the presence of sucrose; and (iii) collecting mutans from the culture Includes.

[0015] GtfJ is one of the genes encoding glucosyltransferases obtained from Streptococcus salivarius ATCC25975, and synthesizes glucans consisting mainly of α(1→3)-linked glucosyl residues (Christine L., et al., Microbiology (1995), 141, 1451-1460).

[0016] GtfJ may be a mutant thereof. Such a mutant has an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of wild-type GtfJ, and has a predetermined activity. Specifically, the GtfJ mutant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity or homology to the amino acid sequence of wild-type GtfJ and has the activity of forming a glycosidic bond. The "identity" of two amino acid sequences refers to the proportion of identical amino acid residues appearing at corresponding positions when the two amino acid sequences are aligned, and the "homology" of two amino acid sequences refers to the proportion of similar amino acid residues appearing at corresponding positions when the two amino acid sequences are aligned. This can be obtained by appropriately aligning the two amino acid sequences to be compared, determining the identical residues present in each sequence, determining the number of matching positions, dividing the number of matching positions by the total number of residues in the sequence to be compared, and multiplying the obtained number by 100. For example, this can be determined using the BLAST (Basic Local Alignment Search Tool) program (Altschul et al., J. Mol. Biol., (1990), 215(3):403-10) or the like.

[0017] The gene encoding GtfJ can be easily obtained by well-known techniques such as PCR using, for example, genomic DNA or cDNA derived from Streptococcus salivarius ATCC25975 as a template. The gene encoding GtfJ may be a mutant. Such a mutant has a nucleotide sequence in which one or more bases have been deleted, substituted, or added to the nucleotide sequence of the wild-type gene. Specifically, a mutant of the gene encoding GtfJ has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity or homology to the nucleotide sequence of the gene. The "identity" of two base sequences refers to the ratio of identical bases appearing at corresponding positions when the two base sequences are aligned, and the "homology" of two base sequences refers to the similarity or correlation between the two base sequences. This can be determined, for example, using the BLAST (Basic Local Alignment Search Tool) program (Altschul et al., J. Mol. Biol., (1990), 215(3):403-10).

[0018] The sucrose non-utilizing bacteria of the present invention are not particularly limited as long as they are microorganisms that do not use sucrose as a nutrient source, and examples thereof include hydrogen-oxidizing bacteria (also referred to as "hydrogen bacteria"). "Hydrogen-oxidizing bacteria" refers to bacteria that oxidize free hydrogen and use the energy generated by the reaction to perform carbon dioxide assimilation. Examples of hydrogen-oxidizing bacteria include Alcaligenes bacteria, such as Larnitonia strains such as Larnitonia eutropha, and Hydrogenovibrio bacteria, such as Hydrogenovibrio marinus. Among these, Larnitonia eutropha is preferred because it can grow even when gaseous carbon dioxide or carbonate is used as the sole carbon source, its entire genome has been analyzed, and a genetic recombination method has been established (Cramm, R. et al., J. Mol. Microbiol. Biotechnol., 16, 38-52 (2009)). Examples of such strains include Larnitonia eutropha H16 (ATCC17699) and Larnitonia eutropha PHB, which lacks the gene (phaC) responsible for polyhydroxyalkanoic acid (PHA) synthesis. - 4 strain (DSM541) (Schlegel HG, Lafferty R. and Krauss I. (1970) The isolation of mutants not accumulating poly-beta-hydroxybutyric acid. Arch. Microbiol. 71: 283-294. http: / / www.ncbi.nlm.nih.gov / pubmed / 4097070).

[0019] In the production of mutan according to the present invention, fructose is produced as a by-product. This fructose can be used as a carbon source for the growth of microorganisms and the production of specific polymers (e.g., polyhydroxyalkanoic acid (PHA)). PHA is a polyester that accumulates intracellularly in microorganisms. In recent years, PHA has attracted attention not only as a biodegradable plastic material but also as a biomass-derived plastic material, and PHAs composed of various polymerizable components depending on the properties of the plastic material have been reported (e.g., Patent No. 5396639). For example, when Larnitonia eutropha H16 strain is used as a sucrose-non-utilizing bacterium, PHA can be produced in a single culture system using the by-produced fructose as a carbon source in addition to the target mutan. Therefore, when producing only mutan, Larnitonia eutropha PHB can be used. - When using the four strains to produce PHA in addition to mutan, Larnitonia eutropha H16 strain can be used.

[0020] The amount of sucrose added is about 0.1 to about 20% by weight based on the total weight of the culture medium, taking into account the amount of mutan produced and the growth of the host microorganism. If the amount is less than 1% by weight, sufficient mutan will not be produced, and even if more than 20% by weight is added, it is difficult to obtain an effect greater than that obtained when 20% by weight is added. The amount of sucrose added is preferably about 1 to about 10% by weight, more preferably about 1 to about 5% by weight, more preferably about 1 to about 3% by weight, more preferably about 1 to about 2% by weight, and particularly preferably about 1% by weight.

[0021] The culture temperature is a temperature at which the bacteria can grow, preferably 15 to 40°C, particularly preferably 20 to 40°C, and even more preferably 28 to 34°C. The culture time is not particularly limited, but for example, 1 to 7 days is preferred for batch culture, and continuous culture is also possible. The culture medium is not particularly limited as long as it can be used by the host of the present invention. The culture medium may further contain a nitrogen source, inorganic salts, other organic nutrient sources, etc. Examples of nitrogen sources include ammonia, ammonium salts such as ammonium chloride, ammonium sulfate, and diammonium hydrogen phosphate, peptone, meat extract, and yeast extract. Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium hydrogen phosphate, magnesium sulfate, and sodium chloride. Other organic nutrient sources include, for example, amino acids such as glycine, alanine, serine, threonine, and proline; vitamins such as vitamin B1, vitamin B12, biotin, nicotinamide, pantothenic acid, and vitamin C; and the like.

[0022] Because mutan is water-insoluble, the mutan of the present invention can be easily recovered from the bacterial cells by centrifugation after the completion of cultivation. In some cases, the bacterial cells can be separated from the culture medium using a centrifuge or the like, dried, and then an organic solvent such as ethanol, methanol, or acetone can be added to the dried bacterial cells to recover lower molecular weight mutan. The resulting precipitate can be washed with water to remove impurities such as by-product fructose, and then stored as a dry powder using an appropriate method such as freeze-drying or vacuum drying. Alternatively, mutan can be recovered from the bacterial cells by the following method. For example, mutan-containing bacterial cells can be dispersed in an aqueous solvent containing a surfactant or the like, disrupted using an ultrasonic disrupter or high-pressure homogenizer, and the insoluble components can then be recovered by filtration or centrifugation. The recovered insoluble components can be washed with an appropriate solvent such as water and dried to recover mutan. Even when PHA is produced using by-product fructose as a carbon source, the method for recovering PHA is similar to the above-mentioned method for recovering mutan, in which the bacterial cells are dispersed in an aqueous solvent containing a surfactant, etc., disrupted using an ultrasonic disrupter or high-pressure homogenizer, and the insoluble components are recovered by filtration or centrifugation. The obtained mutan and PHA can be analyzed by, for example, gas chromatography, nuclear magnetic resonance spectroscopy, or the like.

[0023] A second embodiment of the present invention relates to a sucrose-non-assimilating bacterium into which a gene encoding glucosyltransferase (GtfJ) has been introduced. A preferred example of such a sucrose-non-assimilating bacterium is a Ralstonia eutropha strain. Also preferred is a modified Ralstonia eutropha strain into which the gene has been introduced and in which the gene encoding polyhydroxyalkanoate (PHA) synthase has been deleted. The GtfJ-encoding gene, the sucrose-non-assimilating bacterium, the Ralstonia eutropha strain, the Ralstonia eutropha strain lacking the gene encoding PHA synthase, and the methods for producing these recombinant strains are as described in the first embodiment of the present invention.

[0024] The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples in any way. [Example]

[0025] material 1. Culture Medium (1) NR medium: 2.0 g / L yeast extract, 10 g / L bactotryptone, 10 g / L bonito extract. (2) MS medium: 9.0 g / L NaHPO 4· 12H2O, 1.5g / L KH2PO 4、 0.5g / L NH4Cl, 0.2g / L MgSO 4· 7H2O, 1mL trace elements solution. (3)Trace elements solution: in 0.1N HCl, containing 0.218g / L CoCl2·6H2O, 20.5g / L FeCl3·6H2O, 7.8g / L CaCl2, 0.118g / L NiCl2·6H2O, 0.105g / L CrCl3·6H2O, 0.156g / L CuSO4·5H2O. 2. Strains (1) Ralstonia eutropha H16 (2) Ralstonia eutropha PHB -4(DSM541) Ralstonia eutropha PHB - 4 (DSM541) is a mutant strain of R. eutropha H16, which has a mutation inserted into the gene (phaC) involved in the synthesis of polyhydroxyalkanoic acid (PHA), and is therefore unable to synthesize PHA.

[0026] Example 1 Construction of pBBREE"32d13dPB_gtfJ vector (1) Plasmid pEX-A2J2_gtfJ We commissioned Eurofins to design a gene based on the amino acid sequence of GtfJ (SEQ ID NO: 2) in accordance with the codon usage frequency of E. coli, and to create an artificial gene (SEQ ID NO: 1) by adding NdeI and XhoI sites to the 5' and 3' ends of the gene, respectively. The gene was obtained as the plasmid pEX-A2J2_gtfJ (7.2 kb).

[0027] DNA sequence of gtfJ (SEQ ID NO: 1) catATG (" catATG= "NdeI site"; "ctcgag = XhoI site"; "caccaccaccaccaccac = His tag sequence"; "tga = stop codon")

[0028] Amino acid sequence of GtfJ (SEQ ID NO: 2)

[0029] (2) Plasmid pBBREE”32d13dPB_gtfJ Plasmids pBBREE"32d13dPB_NSDG (7.5 kb) (Tsuge T, et al., FEMS Microbial Lett 277 (2007) 217-222) and pEX-A2J2_gtfJ were treated with restriction enzymes NdeI and BamHI, gel extracted, and then ligated to obtain the phaC fragment of pBBREE"32d13dPB. NSDG The site was replaced with a gtfJ site to generate the plasmid pBBREE"32d13dPB_gtfJ (10.2 kb) (Figure 1).

[0030] Example 2: Preparation of Mutan (1) Preculture R. eutropha PHB - R. eutropha H16 / gtfJ and R. eutropha H16 / gtfJ were cultured in NR medium supplemented with kanamycin (50 μg / mL) at 37°C. Next, using a platinum loop, one colony formed on the plate was inoculated into NR medium supplemented with kanamycin (1.7 mL x 4) and cultured overnight at 37°C and 170 rpm. (2) Main culture The pre-culture solution (1 mL) was added to sucrose-added kanamycin-added MS medium (100 mL), and the mixture was cultured at 30°C and 130 rpm for 72 hours with shaking. 1 mL of the culture solution was collected at 0, 12, 24, 36, 48, and 72 hours after the start of the culture, and the bacterial growth was measured at 600 nm using a spectrophotometer (UV-1900i, Shimadzu Corporation). Pure water was used as a control, and the OD 600 was measured (Figure 2). As shown in Figure 2, R. eutropha PHB without the gtfJ gene - 4 was unable to grow in the presence of 1% sucrose, but the gtfJ-expressing strain grew almost as well as the positive control (fructose medium). After culturing, R. eutropha H16 / gtfJ cells were harvested and dried, and the amount of poly(3-hydroxyalkanoic acid) accumulated was measured by gas chromatography. The result showed that 54 mg / L of polyhydroxybutyric acid had accumulated.

[0031] Gas chromatography (GC): The intracellular PHA content and composition were determined by gas chromatography (Braunegg et al., 1978). First, 10–15 mL of the dried cells were weighed into a pressure-resistant glass tube. 2 mL of sulfuric acid / methanol solution (15 vol% sulfuric acid:85 vol% methanol) and 2 mL of chloroform were added, sealed, and heated in a heat block at 100°C for 140 minutes to induce methanolysis. The sample was stirred approximately every 30 minutes during heating. After cooling to room temperature, 1 mL of purified water was added and vigorously stirred. After standing, the lower layer (chloroform layer) separated into two layers. The lower layer (chloroform layer) was aspirated with a Pasteur pipette and filtered through a 0.45 μm Millex-FH PVDF filter (Millipore). 500 μL of the filtered chloroform layer was mixed with 500 μL of 0.1 vol% methyl caprylate (internal standard) to prepare the sample. The GC system used was a Shimadzu GC-2014s gas chromatograph, and the column used was a GL Sciences NEUTRA-BOND-1 (internal diameter 30 m × 0.25 mm, film thickness 0.4 μm). He and N were used as carrier gases, and a hydrogen flame ionization detector was used to detect the components.

[0032] In addition, under the following conditions, 1 mL of pre-culture solution was added to 100 mL of kanamycin-supplemented MS medium supplemented with sucrose, and cultured at 30°C and 130 rpm for 214 hours with shaking. 1 mL of culture solution was sampled at 0, 24, 77, 103, 166, 190, and 214 hours after the start of culture, and bacterial growth was measured at 600 nm using a spectrophotometer (UV-1900i, Shimadzu Corporation). Pure water was used as a control, and OD 600 was measured (Figure 3).

[0033] [Table 1]

[0034] (3) Verification of sucrose consumption in high-performance liquid chromatography Sucrose consumption during the cultivation was confirmed by high-performance liquid chromatography (apparatus: Shimadzu Prominence (Shimadzu Corporation); column: Amide-80; column temperature: 65°C; eluent: 70% acetonitrile aqueous solution; flow rate: 0.6 mL / min) (Figure 4).

[0035] (4) Bacteria collection / freeze drying The bacterial suspensions No. 1 and No. 4 in Table 1 were each transferred to a 50 mL Falcon tube and centrifuged (4000 rpm, 10 minutes, 25°C). The supernatant was discarded, and approximately 30 mL of pure water was added and suspended. Centrifugation was performed under the same conditions. The supernatant was discarded, and approximately 30 mL of pure water was added and suspended. The supernatant was discarded, and the pellet was placed in a -80°C freezer and frozen. After confirming that it was frozen, the pellet was freeze-dried for several days to obtain dried bacterial cells.

[0036] (5) Mutan Refining Approximately 1.5 g of dried cells No. 1 from Table 1 and 20 mL of 1M NaOH aqueous solution were added to a glass test tube (approximately 100 mL capacity) for automated synthesizers (containing a football-shaped stirrer). The tube was heated to 100°C and held at 100°C for 1 hour. The tube was then left overnight to cool to room temperature. 40 mL of methanol was added, and the mixture was left to stand for approximately 1 day, yielding a brown precipitate. The supernatant was removed by centrifugation (4000 rpm, 10 minutes, 25°C), and approximately 30 mL of 75% aqueous methanol was added to suspend the precipitate. After centrifugation under the same conditions, the precipitate was dissolved in approximately 10 mL of 1M NaOH and filtered through No. 2 filter paper. The mixture was further filtered through No. 5 filter paper, and approximately twice the volume of methanol was added to the filtrate to insolubilize it. The process from filtration through No. 5 filter paper to insolubilization was repeated two more times. The insolubilized material was centrifuged to obtain a precipitate (125 mg of mutan), which was then freeze-dried. In addition, the same procedure as above was carried out using dried cells of No. 4 in Table 1 to obtain a precipitate (125 mg of mutan) which was then freeze-dried.

[0037] (6)NMR analysis The freeze-dried precipitate obtained in (5) was dissolved in NaOH (solid) and filtered through a 0.45 μm filter. 13 The structure of the precipitate was confirmed by C NMR (BioSpin Avance III HD 500) analysis. of measured precipitate 13 C NMR was in good agreement with authentic samples, supporting the structure of α-1,3-glucan. Mutan (preparation): 13 C-NMR: (500MHz:D2O): δ61.3(C6), δ70.9(C4), δ71.4(C2), δ73.2(C5), δ84.7(C3), δ101.6(C1). Mutan (product): 13 C-NMR: (500MHz:D2O): δ61.7(C6), δ71.1(C4), δ71.7(C2), δ73.4(C5), δ84.1(C3), δ101.1(C1). [Industrial Applicability]

[0038] The production method of the present invention can be used for industrial production of mutan.

Claims

1. (i) introducing a gene encoding glucosyltransferase J (GtfJ) into a sucrose-non-utilizing bacterium to prepare a GtfJ-expressing strain; (ii) culturing the expression strain in the presence of sucrose; and (iii) collecting mutans from the culture A method for producing mutan, comprising:

2. The method according to claim 1, wherein the sucrose non-utilizing bacteria are hydrogen-oxidizing bacteria.

3. The method according to claim 2, wherein the hydrogen-oxidizing bacterium is a Ralstonia eutropha strain or a modified Ralstonia eutropha strain obtained by deleting a gene encoding polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha.

4. The method according to claim 3, wherein the modified Ralstonia eutropha strain is used as the hydrogen-oxidizing bacterium and only mutan is produced.

5. The method according to claim 3, wherein a Ralstonia eutropha strain is used as the hydrogen-oxidizing bacterium, and PHA is further produced using fructose produced as a by-product in the step (ii) as a carbon source.

6. The method according to any one of claims 1 to 5, wherein the amount of sucrose added is 0.1 to 20% by weight.

7. A Ralstonia eutropha strain into which a gene encoding glucosyltransferase (GtfJ) has been introduced, or a Ralstonia eutropha strain into which the gene has been introduced and from which the gene encoding polyhydroxyalkanoate synthase has been deleted.

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