JP2012044868A - High-temperature-resistant bacterium producible of 5-ketogluconic acid, and method for producing 5-ketogluconic acid using the high-temperature-resistant bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a high-temperature-resistant bacterium which can be applied to industrial production of 5-keto-D-gluconic acid.SOLUTION: There are provided a high-temperature-resistant bacterium which belongs to the genus Gluconobacter and can produce 5-keto-D-gluconic acid at 30 to 38°C, or the high-temperature-resistant bacterium which is a mutant strain produced by disrupting an FAD-GADH gene; and a method for producing 5-keto-D-gluconic acid, in which the 5-keto-D-gluconic acid is produced by culturing the high-temperature-resistant bacterium at 30 to 37°C in the presence of PQQ and/or a Ca ion.

Description

  The present invention relates to a thermotolerant bacterium having an ability to produce 5-ketogluconic acid (5KGA) at a high temperature, and a method for producing 5KGA using the thermotolerant bacterium.

  Gluconobacter is a type of acetic acid bacterium that can oxidize a wide range of compounds such as sugars, sugar alcohols, and sugar acids, and can accumulate the oxidized products in a medium with high yield. Enzymes involved in these oxidation reactions are D-glucose, D-sorbitol, D-mannitol, glycerol, gluconic acid and ketogluconic acid dehydrogenase. All of these enzymes are tightly bound to the surface layer of the cytoplasmic membrane, and the electrons extracted from the substrate are transferred to the terminal ubiquinone oxidase through ubiquinone, and bioenergy is generated along with the electron transfer reaction.

  It is known that the oxidation of D-glucose to keto-D-gluconic acid is catalyzed by enzymes present outside the cell membrane. D-glucose is oxidized to glucono-δ-lactone by membrane-bound pyrroloquinoline quinone (PQQ) -glucose dehydrogenase, and glucono-δ-lactone is converted to D-gluconic acid by gluconolactonase. . Matsushita et al. Report that the formation of ketogluconic acid observed in Gluconobacter is catalyzed by two types of membrane-bound gluconate dehydrogenase (Non-patent Document 1). One is FAD-containing 2-ketogluconic acid producing enzyme, that is, FAD-gluconate dehydrogenase (FAD-GADH), and the other is PQQ-containing 5-keto-D-ketogluconic acid producing enzyme, In other words, it is PQQ-glycerol dehydrogenase (PQQ-GLDH) (FIG. 1). In addition, it is reported by Shinagawa et al. That FAD-GADH has three subunits (Non-patent Document 2). The three subunits include FAD-containing dehydrogenase subunits and three-heme-containing cytochromes. C subunit, and unknown small subunit. It has been clarified by Matsushita et al. That the latter enzyme that catalyzes 5KGA production is the same as the PQQ-containing polyol dehydrogenase (Non-Patent Document 3). PQQ-GLDH is a D-arabitol dehydrogenase, It is known as an oxidase of various sugar alcohols such as D-sorbitol dehydrogenase or glycerol dehydrogenase. PQQ-GLDH can react with various substrates, but has high stereospecificity in which the reaction is catalyzed according to the so-called “Bertrand-Hudson method”. This enzyme can oxidize only the C5 position of D-gluconic acid in order to produce 5KGA from D-gluconic acid, but the enzyme affinity with D-gluconic acid is low. The gene for this enzyme was cloned from Gluconobacter suboxydans IFO 3255 and two open reading frames were found. One is presumed to encode a hydrophobic region protein of the 5-transmembrane region, and the other encodes a dehydrogenase subunit similar to several PQQ-dependent enzymes, particularly membrane-bound glucose dehydration. Similar to the PQQ domain of elementary enzymes. On the other hand, 2KGA reductase and 5KGA reductase located in the cytoplasm function to convert and assimilate 2KGA and 5KGA into D-gluconic acid after being transported to the cytoplasm by transport proteins.

  5KGA is a useful substance that is a raw material for tartaric acid, xylic acid, vitamin C, and important fragrance compounds (Non-patent Document 4). Grey et al. Provide a method for producing vitamin C using 5KGA, which is a method that can be industrially produced under milder conditions than the Reichstein method that is industrially performed today (Patent Document 1). 2).

Most Gluconobacter strains produce 2KGA and 5KGA from D-glucose. Thus, 5KGA by Gluconobacter bacteria produces 2KGA as a major byproduct, and the production of the two ketogluconic acids competes in vivo. The present inventors have reported a method of selecting Gluconobacter bacteria that specifically produce 5KGA having a conversion rate of 80% or more from a wild strain with respect to a D-glucose substrate (Patent Document 3). Moreover, since 2KGA production is catalyzed by the FAD-GADH gene, a FAD-GADH-deficient mutant of Gluconobacter oxydans 621H (identical to Gluconobacter suboxydans IFO12528) is prepared, and D-glucose is used. There is a report that 5KGA was produced almost specifically (Non-patent Document 5). However, the optimum temperature for the production of 5KGA using this cold strain is around 20 ° C. In an industrial scale culture process, a cooling device is required to reduce the increase in culture temperature. From the viewpoint of cost reduction, a strain that produces 5KGA at a higher temperature is desired. The present inventors reported the gluconobacter genus microbe which has high temperature tolerance about the strain obtained from various materials in Thailand (nonpatent literature 6). However, the high 5KGA production strain is not disclosed.
Matsushita, K .; , Et al, 1994. p. 247-301. In D. W. T.A. A. H. Rose (ed.), Advance In Microbiology Pyology, vol. 36. Academic Press Ltd. , London Shinagawa, E .; , Et al, Agricultural Biological Chemistry 48: 1517-1522, 1984. Matsushita, K .; , Et al, Applied and Environmental Microbiology 69: 1959-1966, 2003. Salusjarvi, T .; , Et al, Applied Microbiology and Biotechnology 65: 306-314, 2004. Elfari, M .; , Et al, Applied Microbiology and Biotechnology 66: 668-674, 2005. Moonangmee, D.M. , Et al, Bioscience, Biotechnology and Biochemistry 64: 2306-2315, 2000. U.S. Pat. No. 2,421,621 U.S. Pat. No. 2,421,612 JP 2008-237191 A

  The main object of the present invention is to provide high-temperature resistant bacteria that can be applied to industrial 5KGA production.

  The present inventors isolated three novel high-temperature-resistant Gluconobacter species by screening strains that can grow at a high temperature of 37 ° C. and cannot produce 5KGA. Furthermore, the present invention was completed by finding that 5KGA can be produced specifically by disrupting the FAD-GADH gene system.

  That is, the present invention provides the following (1) to (5).

  (1) A thermotolerant bacterium belonging to the genus Gluconobacter and having 5KGA producing ability at 30 to 38 ° C., or a mutant strain thereof, having 5KGA producing ability.

  (2) A thermotolerant bacterium in which the mutant strain described in (1) above is a mutant strain in which the FAD-GADH gene is disrupted.

  (3) A plasmid in which a kanamycin cassette is inserted into a PCR product obtained by amplifying a DNA fragment having the nucleotide sequence shown in SEQ ID NO: 7, 8, or 9 from a mutant strain in which the FAD-GADH gene is disrupted is introduced into the cell. The thermotolerant bacterium according to (2) above, which is a strain constructed by introduction and homologous recombination.

  (4) Gluconobacter fratheuri THE42 strain (Accession number NITE-P-655), Gluconobacter fratheuri THG 42 strain (Accession number NITE-P-657), Gluconobacter fratheuri THF55 strain (Accession number NITE- P-659), Gluconobacter frateuri THE42 gndG :: Km strain (Accession number NITE-P-656), Gluconobacter frateuri THG42 gndG :: Km strain (Accession number NITE-P-658), Gluconobacter・ Frateuri THF55 gndG :: Km strain (Accession No. NITE-P-660), one or more thermotolerant bacteria having the ability to produce 5KGA.

  (5) The high-temperature resistant bacterium belonging to the genus Gluconobacter according to any one of (1) to (4) above is cultured at 30 to 37 ° C. in the presence of PQQ and / or Ca ions to produce 5KGA. A method for producing 5KGA, wherein 5KGA is collected.

  According to the present invention, 5KGA, which is a raw material for useful industrial products, can be cultured under high temperature conditions, and in order to maintain an appropriate temperature during the culture, the high cooling cost required for psychrophilic bacteria is reduced. can do. Further, since the mutant strain specifically produces 5KGA, the purification process is easy and industrially advantageous.

  The high-temperature-resistant bacteria of the present invention are Gluconobacter bacteria that can produce 5KGA at a high temperature of 30 to 38 ° C. Examples of the genus Gluconobacter include Gluconobacter suboxydans, Gluconobacter oxydans, Gluconobacter frateuri, and the like. It may be a mutant strain obtained by transforming a wild strain.

  The thermotolerant bacterium of the present invention can be obtained by the following screening method. That is, as an initial screening, one colony is inoculated into a glucose-gluconic acid medium from a high temperature-resistant Gluconobacter bacterium stored in an appropriate medium, for example, a potato agar medium, and cultured at 30 ° C. to 37 ° C. for 2 to 3 days. The strain that produced a brown compound in the medium is excluded as a 2,5-diketogluconic acid (2,5DKGA) producing strain. 2,5DKGA is a 2KGA oxidation product catalyzed by membrane-bound FAD-2-ketogluconate dehydrogenase present in some Gluconobacter species. Strains that produce a large amount of 2,5DKGA form a large amount of 2KGA and the formation of 5KGA is expected to be low, so it is desirable to exclude those strains. The remaining bacteria are further cultured in a glucose-gluconic acid medium at 30 to 38 ° C., and 5KGA producing strain is selected by quantifying 5KGA in the culture solution. The production amount of 5KGA can be examined, for example, by a color development reaction with a resorcinol reagent that develops color by a reduction reaction.

  The obtained high temperature resistant Gluconobacter wild strain has been deposited in Japan. The accession number is the Gluconobacter frateuri THE42 strain, the accession number NITE-P-655, the Gluconobacter frateuri THG42 strain is the accession number NITE-P-657, and the Gluconobacter frateuri THF55 strain is the accession number. NITE-P-659.

  The mutant strain of the genus Gluconobacter having high temperature resistance of 5KGA of the present invention can be selected not only from the above wild strain but also from the 2,5DKGA production strain as a parent strain and involved in the formation of the parent strain 2KGA. It has been modified to specifically produce 5KGA by disrupting the FAD-GADH gene, which is an enzyme that performs The defective strain in which the FAD-GADH gene is disrupted is normal by deleting the FAD-GADH gene on the chromosome of the wild strain or a part of its DNA sequence or inserting a drug resistance gene into the gene. By transforming a bacterium with a DNA containing a gene modified so as not to produce a functional enzyme, and causing homologous recombination with a normal gene on the chromosome, the gene on the chromosome is destroyed. To make. Such gene disruption by homologous recombination gene replacement has already been established as a method using linear DNA or plasmid. Preparation of the FAD-GADH gene disruption strain of the high temperature tolerance Gluconobacter genus bacteria of this invention can be performed with the following operation methods and procedures.

  1) Extract DNA from high temperature resistant Gluconobacter spp. Genomic DNA extraction method from high temperature resistant Gluconobacter genus is described in the method of Sambrook et al. (Sambrook, J., et al., 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Label LaborlaborLaborLaboratoryLaboratoryLaboratoryLaboratory , NY)).

  2) The target FAD-GADH gene is cloned. Cloning of the FAD-GADH gene can be carried out by PCR using primers designed from the region maintained in the dehydrogenase subunit in the gene for genus Gluconobacter or Erwinia GADH, FAD- Although it can be performed by hybridization using the GADH gene, the most efficient method is to use a primer designed based on the sequence of the FAD-GADH gene reported in the genome of Gluconobacter oxydans 621H. Using the genomic DNA obtained in 1) as a template, PCR can be performed, and a gene containing the full length of the FAD-GADH gene can be amplified and obtained by PCR. The entire sequence of the cloned gene can be obtained from GENETYX-MAC (Software Development, Tokyo, Japan) or Clone Manager (Scientific and Educational Software) using ABI PRISM 310 (manufactured by PE Biosystems). To obtain sequence data. In addition, homology search and alignment analysis are performed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) and CLUSTAL W (www.ebi.ac.uk/clustalw), respectively. Obtainable.

  Examples of the DNA fragment obtained by cloning the FAD-GADH gene of the present invention include gndF (SEQ ID NO: 7), gndG (SEQ ID NO: 8), gndH (SEQ ID NO: 9) and the like. These DNA fragments can be amplified and used as a plasmid for FAD-GADH gene disruption.

  3) A FAD-GADH gene disruption plasmid is prepared based on the sequence information of the cloned gene. The FAD-GADH gene disruption plasmid was prepared by incorporating the PCR-amplified FAD-GADH gene into an E. coli vector such as pGEM-T Easy Vector that cannot replicate in the high temperature resistant Gluconobacter genus. It can be prepared by inserting a cassette made of a drug resistant substance such as kanamycin into the restriction enzyme site of DNA.

  4) Next, a FAD-GADH gene-disrupted strain is prepared by introducing a plasmid. The FAD-GADH gene disruption strain was prepared by subjecting the plasmid for gene disruption to an electric pulse method (Agric. Biol. Chem., 54: 443-447, 1990, Res. Microbiol. 144: 181-185, 1993) and plasmids are introduced into cells by a highly efficient gene transfer method such as conjugation, and the FAD-GADH gene is disrupted (or inactivated) by homologous recombination to the chromosome. Can be performed. Confirmation of the disruption of the FAD-GADH gene is performed by applying a strain in which a drug resistance gene fragment is inserted into a chromosome onto a plate medium containing an appropriate concentration of the drug, and transforming it to a high temperature resistant Gluconobacter genus. This can be done by selecting the fungus and confirming that the selected bacterial strain has lost the enzyme activity for 2KGA production.

  The transformed high temperature tolerant Gluconobacter bacterium of the present invention prepared as described above is prepared by using a suitable medium such as potato agar medium (20 g glycerol, 5 g glucose, 10 g yeast extract, 10 g polypeptone, 2 g agar, 100 ml). Can be stored at a low temperature of 0 to 5 ° C. Long-term storage can be performed at −80 ° C. using a potato medium containing 50% glycerol.

  Examples of the mutant strain of the transformed high temperature resistant Gluconobacter genus of the present invention include the following bacteria. A mutant strain of Gluconobacter frateuri THE42 strain (Accession No. NITE-P-655) is a Gluconobacter frateuri THE42 gndG :: Km strain and has been deposited in Japan as Accession No. NITE-P-656. The mutant strain of Gluconobacter frateuri THG 42 (accession number NITE-P-657) is Gluconobacter frateuri THG42 gndG :: Km strain, which was similarly deposited as accession number NITE-P-658. In addition, the mutant strain of Gluconobacter frateuri THF55 (accession number NITE-P-659) is Gluconobacter frateuri THF55 gndG :: Km, and has been deposited in the same manner as accession number NITE-P-660. Yes.

  In the production of 5KGA of the present invention, the wild strain or mutant strain of the high temperature-resistant Gluconobacter genus of the present invention can be grown and cultured under aerobic conditions, and 5KGA can be produced and accumulated in the medium.

  The culture of the high temperature resistant Gluconobacter genus of the present invention can be performed using a normal nutrient medium containing a carbon source, a nitrogen source, an inorganic salt, and the like. Examples of the carbon source include monosaccharides such as glucose, galactose, fructose and mannose, disaccharides such as lactose, sucrose and maltose, gluconates such as sodium gluconate and potassium gluconate, glycerin, maltitol, lacriitol, mannitol Sugar alcohols such as sorbitol, xylitol, and arabitol are used. Among these, glucose or gluconic acid is preferable, and glucose is most preferable.

  As the nitrogen source, for example, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea or the like can be used alone or in combination. Further, as the inorganic salt, for example, potassium monohydrogen phosphate, potassium dihydrogen phosphate or magnesium sulfate can be used. In addition, various nutrients such as polypeptone, meat extract, yeast extract, potato extract, and corn steep liquor can be appropriately added to the medium as necessary.

  In the present invention, a cofactor can be added to the medium for the enzyme involved in the production of 5KGA. It has been clarified that PQQ-GLDH involved in the production of 5KGA of the present invention causes a structural change of the enzyme when a high temperature resistant Gluconobacter bacterium is grown at high temperature. Therefore, the enzyme activity can be maintained at a high level by supplementing the medium with a cofactor. Examples of cofactors include magnesium ions, calcium ions, ammonium ions, sodium ions, and potassium ions. These ions include magnesium chloride, magnesium carbonate, calcium chloride, calcium carbonate, ammonium carbonate, sodium carbonate, and potassium carbonate. Compounds can be added to the medium. Preferred compounds are magnesium chloride, magnesium carbonate, calcium chloride, and calcium carbonate, and most preferred is calcium chloride. The amount of these compounds added to the medium is 1 to 50 mM, preferably 5 mM.

  Furthermore, the coenzyme pyrroloquinoline quinone (PQQ) may be mentioned as a cofactor to be added to the medium for the enzyme activity involved in 5KGA production in the present invention. In order to maintain the enzyme activity, supplementation of the coenzyme to the medium can be performed to increase the production of 5KGA. The addition amount of PQQ is 0.1-100 μM, preferably 1-10 μM is added to the medium.

  Cultivation can usually be performed at a temperature of 28 ° C. to 38 ° C., preferably 30 to 37 ° C. under aerobic conditions such as aeration stirring or shaking. The pH during culture is preferably in the range of about 5 to 7, and pH adjustment during culture can be performed by adding an acid or an alkali. The culture period is 1 to 7 days, preferably 2 to 5 days, to obtain 5KGA. Production of 5KGA is seen in the first growth stage, but the highest production is seen in the stable growth period.

  The recovery of 5KGA from the cultured cells and medium of the high-temperature-resistant Gluconobacter genus of the present invention can be performed according to a general fermentation production method, for example, known methods such as centrifugation, membrane separation, column chromatography, etc. This method can be used.

Hereinafter, examples will be given to describe the present invention in more detail, but the present invention is not limited thereto.

<Screening of high temperature resistant 5KGA production strain of Gluconobacter>

1. Screening of wild strains All Gluconobacter bacteria used in the screening experiments were obtained from various materials in Thailand from Moonangmee, D. et al. (Bioscience, Biotechnology and Biochemistry 64: 2306-2315, 2000), and potato agar medium (20 g glycerol, 5 g glucose, 10 g yeast extract, 10 g polypeptone, 2 g agar, 100 ml potato extract with 1 L of distilled water. And saved. For initial screening, one colony from all isolates was inoculated into 2% glucose-gluconate medium (20 g glucose, 20 g sodium gluconate, 3 g polypeptone, 3 g yeast extract to 1 L with distilled water), 30 ° C. The strains that were cultured for 48 hours at 37 ° C. or 60 hours at 37 ° C. and produced a brown compound in the medium were excluded as 2,5-diketogluconic acid producing strains. Thereafter, 200 μl of the medium and 1 ml of Resorcinol reagent (0.5% Resorcinol 49 ml, concentrated hydrochloric acid 168 ml, distilled water 273 ml) were reacted at 80 ° C. for 20 minutes. The reaction product of the Resorcinol reagent and D-glucose was a red compound, and in the case of 5KGA, a black brownish green precipitate was formed. On the other hand, D-gluconic acid and 2KGA were not found in the confirmed reaction product. In order to obtain the best strain as a high temperature resistant 5KGA producing strain, 24 strains (Table 1) isolated in the initial screening were prepared from potato medium (20 g glycerol, 5 g glucose, 10 g yeast extract, 10 g polypeptone, 100 ml potato extract. 1 μl with distilled water), and 10 μl of the culture solution was inoculated into 1 ml of 2% glucose-gluconic acid medium and cultured at 30 ° C. for 36 hours or 37 ° C. for 48 hours. The culture solution was reacted with the Resorcinol reagent. Furthermore, growth and 5KGA accumulation were compared by measuring the absorbance of the reaction product produced by the reaction.

2.2 Determination of KGA and 5KGA 2KGA and 5KGA were obtained from Saichana. The amount was enzymatically determined according to the method of I et al. (Bioscience, Biotechnology and Biochemistry 71: 2478-2486, 2007). Qualitative analysis of 2KGA and 5KGA was performed by thin layer chromatography analysis. The sample was spotted on a silica gel 60 plate (Merck) and developed with a solvent containing ethyl acetate: acetic acid: methanol: non-ionized water (6: 1.5: 1.5: 1). The plate was dried and sprayed with a color reagent (2 g diphenylamine, 2 ml aniline, 100 ml acetone, 15 ml phosphoric acid pre-use preparation) and heated at 120 ° C. for 10-20 minutes until color developed. 5KGA and 2KGA appeared as dark purple and reddish brown spots, respectively.

3. Screening results Three of the 24 isolates, THE42, THF55, and THG42, showed high production of 5 KGA at both 30 ° C and 37 ° C (Table 1).

The formation of 5KGA by the selected three thermotolerant bacteria was confirmed by TLC in FIG. 2. Regarding the characteristics of the growth and production of the three strains, 2KGA and 5KGA production at 30 ° C. and 37 ° C. Production Gluconobacter suboxydans IFO12528 bacteria were compared and examined. The results are shown in Table 2. The three thermotolerant bacteria were the same in both growth and production of 2KGA as the main product. After culturing at 30 ° C. for 48 hours, the thermophilic bacteria showed lower growth, lower 2KGA production, and higher 5KGA production than the thermotolerant bacteria. At 37 ° C., the growth of all the bacteria was slower than at 30 ° C., so that the culture time was 72 hours in order to increase 2KGA and 5KGA products. Thermophilic bacteria grew little at 37 ° C, indicating low 2KGA and 5KGA production. Three thermotolerant bacteria at this temperature showed growth and growth at 37 ° C and the possibility of 5KGA production, which was not possible with room temperature bacteria, although growth and production of 2KGA and 5KGA were low compared to 30 ° C. It was.

<Production of mutant strain of high temperature resistant Gluconobacter>
About high temperature tolerance Gluconobacter microbe, the microbe which produces 5KGA specifically was produced. 2KGA is produced by FAD-gluconate dehydrogenase (FAD-GADH). In isolated high temperature resistant Gluconobacter bacteria that compete with 5KGA and produce 2KGA as the main product, 2KGA production was eliminated by disrupting the FAD-GADH gene. The primers used for the following cloning are shown in Table 3.

1. Cloning of FAD-gluconate dehydrogenase gene from thermotolerant Gluconobacter fungus FAD-GADH structural gene has been reported in the genomic sequence of Gluconobacter oxydans 621H (Nature Biotechnology 23: 195-200, 2005). ). It is composed of three subunits (dehydrogenase subunit, cytochrome subunit, and small subunit). A gndL-F primer (SEQ ID NO: 1) and a gndL-R primer (SEQ ID NO: 2) designed based on the FAD-GADH gene sequence were used as PCR primers. This sequence is the same as Elvinia Cypripedi (Journal of Bacteriology 179: 6566-6572, 1997), Gluconobacter dioxyacetonicus IFO 3271 (Microbiology 73: 6551-6556, 2007), Gluconobacter oxydane 621H (Naturol 23: 195-200, 2005) was designed from the region conserved in the dehydrogenase subunit in the GADH gene. This primer pair was subjected to PCR using PuReTaq Ready-To-Go PCR Beads kit (manufactured by GE Healthcare) using the genomic DNA of each thermotolerant bacterium as a template. For PCR, mGeneAmpPCR System 2400 (manufactured by Perkin Elmer) was used, and the PCR temperature cycle was performed as follows. One cycle at 95 ° C. for 30 seconds, followed by 25 cycles of 95 ° C. for 30 seconds, 55 ° C. for 1 minute, and 68 ° C. for 1 minute were maintained at 37 ° C. at the end of all cycles. DNA fragments were separated on an agarose gel and purified by QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) or MagExtractor DNA fragmentation kit (Toyobo, Tokyo), Japan. This resulted in a 525 bp product of PCR. This sequence was found to be homologous to GOX1231, which is a dehydrogenase subunit of the FAD-GADH gene of Gluconobacter oxydans 621H.

In order to obtain the entire sequence of the gene, F-igL-1 (SEQ ID NO: 3) and F-igL-2 (sequence) are specific primer pairs designed from the sequence obtained by sequencing the PCR product. No. 4), PCR was performed by adding ₂ mM MgCl ₂ and 0.5% DMSO to TaKaRa LA Taq ^™ (Takara Bio Inc.) using a recircularized EcoRI digest of genomic DNA as a template. . One cycle at 95 ° C. for 30 seconds, followed by 25 cycles of 95 ° C. for 30 seconds, 55 ° C. for 1 minute, and 68 ° C. for 6 minutes were maintained at 37 ° C. at the end of all cycles. The entire sequences of the dehydrogenase and small subunit genes were prepared by using FacL-1 (SEQ ID NO: 3) and R-igL-2 as primers using a template obtained by recircularization of SacII or EcoRI digest of genomic DNA. Amplified with (SEQ ID NO: 4). The gene for the cytochrome C subunit was obtained from in vitro cloning methods.

  Sequences were identified by sequencing 6 ORF fragments containing 3 ORFs (gndF, gndG, gndH) predicted to be derived from the FAD-GADH gene obtained from each strain and 3 ORFs encoding proteins of unknown function. Sequencing was performed using ABI PRISM 310 (PE Biosystems). The sequence data was analyzed using GENETYX-MAC (manufactured by Software Development) or Clone Manager (manufactured by Scientific and Educational Software). Homology search and alignment analysis were performed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) and CLUSTAL W (www.ebi.ac.uk/clustalw), respectively. . As an example, a fragment map obtained from the THF55 strain is shown in FIG. ORF-1, ORF-2, and ORF-3 showed homology to proteins of unknown function encoded by GOX1234, GOX1233, and GOX1229 shown in the genome of Gluconobacter oxydans 621H. The three ORFs of gndF, gndG, and gndH had 76%, 89%, and 68% identity to GOX1232, GOX1231, and GOX1230, which were confirmed to be FAD-GADH genes in the genome. The sequence obtained in this study is pending application for registration with DNA Data Bank of Japan (DDBJ).

  The determined amino acid sequence of GndF is shown at Berks, B. et al. C. (Molecular Microbiology 35: 260-274, 2000) reported that there is a tat-dependent special signal sequence containing two arginine residues. GndF is also known as a small subunit of FAD-GADH as Toyama. 44% identity to GndS of Gluconobacter dioxyacetonicus IFO3271 revealed by H et al. (Applied and Environmental Microbiology 73: 6551-6556, 2007).

  The determined amino acid sequence of GndG showed a glycine box (GxGxxG), which is a signal for binding of FAD to the N-terminus so that similar sequences can be seen in several FAD-containing enzymes. GndG had a region sequence maintained within the glucose-methanol-choline (GMC) oxidoreductase family, a protein family that binds FAD as a cofactor. In addition, the Toyama. It showed 61% identity with GndL of FAD-GADH dehydrogenase subunit of Gluconobacter dioxyacetonicus IFO3271 found by H et al.

  GndH is similar to many membrane-bound cytochrome c subunits found in Gluconobacter bacteria, such as alcohol dehydrogenase, sorbitol dehydrogenase, and aldehyde dehydrogenase. Three heme c-binding motifs (CxxCH) The sequence is shown. Furthermore, it showed 44% identity to GndC of Gluconobacter dioxyacetonicus IFO3271 (20).

2. Construction of FAD-GADH Dependent Mutant of High Temperature Resistant Gluconobacter genus The template genomic DNA is glucosylated in the logarithmic growth phase according to the method of Marmur (J. Mol. Biol. 3: 208-218, 1961). Isolated from Novacter. A plasmid for disruption of gndG was prepared from E. coli DH5α strain using QIA Prep Spin Miniprep kit (Qiagen). Using the F-gnDH-3 primer (SEQ ID NO: 5) and the R-gnDH-2 primer (SEQ ID NO: 6), the template genomic DNA was amplified to the SmaI site of the 1.9 kb PCR fragment. Was constructed by inserting an EcoRV fragment containing the Kmr cassette (pGEM-CA-F :: Km). Analysis of the restriction enzyme cleavage pattern confirmed that the Kmr cassette was inserted in the same transcription direction as gndG. The prepared plasmid was introduced into Gluconobacter by electroporation, and the introduced cells were cultured in potato medium at 30 ° C. for 6 hours for gene recombination. After 2-3 days, the colonies obtained on SG agar medium (1% sorbitol, 1% glycerol, 0.3% polypeptone, 0.3% yeast extract, 2% agar) containing 50 μg / ml kanamycin were picked up. 1 ml of SG medium (1% sorbitol, 1% glycerol, 0.3% polypeptone, 0.3% yeast extract) containing 50 μg / ml kanamycin was inoculated. Subsequently, mutant strains were inoculated into two new agar media with or without kanamycin 50 μg / ml and ampicillin 50 μg / ml, and kanamycin-resistant colonies that did not show ampicillin resistance were selected. The gene disruption was confirmed by PCR using a primer that amplified by covering the insertion region of the Kmr cassette (FIG. 4). As a result, gndG: Km mutant strains from the THE42, THF55, and THG42 strains were obtained, and all three mutant strains were confirmed to have no 2KGA productivity from TLC analysis (FIG. 5).

3. Confirmation of Mutant by Enzyme Activity Membrane-bound enzyme activity against D-arabitol and glucose in wild and mutant strains was prepared from cells grown at 30 ° C. and 37 ° C. by membrane fractionation, and Toyama. H et al. (Bioscience Biotechnology and Biochemistry 69: 1120-1129, 2005). Membrane-bound enzyme activity was measured by reducing activity using ferricyanide and PMS, and expressed as ferricyanide reductase activity or PMS-DCIP reductase activity (FIGS. 6a and b).

Ferricyanide reductase activity was measured using potassium ferricyanide as an electron acceptor. At 25 ° C., 0.9 ml of a reaction solution containing 0.1 M substrate, McIlvaine buffer (McB, pH 5.0), and an appropriate amount of enzyme solution was incubated for 5 minutes, and then 0.1 M potassium ferricyanate. 0.1 ml of the dough was added and stirred. After incubation for a certain period of time, the reaction was stopped by adding 0.5 ml Dupanol reagent (0.3% Fe ₂ (SO ₄ ) ₃ , 8.1% phosphoric acid, 0.3% SDS). After being allowed to stand for 20 minutes to stabilize the color, the mixture was diluted with 3.5 ml of distilled water so that the total amount of the mixture became 5 ml, and the absorbance at 660 nm was measured with a spectrophotometer U2000 (manufactured by Hitachi, Ltd.). One unit of enzyme activity was defined as the amount of enzyme that oxidized 1 μmol of substrate per minute.

The phenazine mesosulfate (PMS) reducing activity of the membrane fraction was measured at 25 ° C. in 1 ml reaction solution with 100 mM substrate, 0.2 mM PMS, 1.2 mM NaN ₃ , appropriate amount of enzyme, McB (pH 5. 0) was subjected to a coupling reaction with 0.11 mM 2,6-dichlorophenolindophenol (DCIP), and a blue fading of 600 nm was measured with a spectrophotometer U2000 (manufactured by Hitachi, Ltd.). One unit of enzyme activity was the amount of enzyme that oxidized 1 μmol of substrate per minute in this assay system.

  As shown in FIG. 6a, the D-arabitol dehydrogenase activity of the wild strain and the mutant strain grown at 30 ° C. was the same, but the D-gluconate dehydrogenase activity was 30% of that of the wild strain. More than that. The decrease in activity was due to inactivation of the gndG gene encoding the FAD-GADH dehydrogenase subunit, and the PQQ-GLDH activity in response to D-gluconic acid was maintained as it was. In FIG. 6b, the membrane-bound enzyme activity from cells grown at 37 ° C. in the wild strain and the mutant strain is lower than that at 30 ° C., and the D-arabitol dehydrogenase activity of the mutant strain is the same as that in the wild strain. However, it was shown that the D-gluconate dehydrogenase activity in the mutant was reduced in the same manner as at 30 ° C. In addition, at 37 ° C., the addition of PQQ increased the activity by a factor of 2 or more in both the wild strain and the mutant strain. This indicates that PQQ necessary for PQQ-GLDH is insufficient when the bacteria grow at high temperature.

<Production of 5KGA by FAD-GADH-deficient mutant>
Wild strains and mutant strains were cultured in 2% glucose-2% gluconic acid medium at 30 ° C. and 37 ° C., and the production amounts of 2KGA and 5KGA were measured along the culture time (FIGS. 7a and 7b). The growth of the wild strain at 37 ° C. was slightly lower than 30 ° C., 2KGA was produced as the main product at both 30 ° C. and 37 ° C., and the production amount was more than twice that of 5KGA. Production of the two products was seen from the first growth stage, while the highest production was seen in the stationary growth phase. At 30 ° C, a decrease in 2KGA and 5KGA was seen after 60 hours of culture, but this was not seen in culture at 37 ° C. When the production of 5KGA by the gndG :: Km mutant strain was compared with that of the wild strain, all of the three mutant strains were mainly 5KGA production and only 2KGA production was slight at 30 ° C. The highest production of 2KGA and 5KGA was seen after 60 hours, similar to the wild type. Even at 37 ° C., the mutant strains still produced mainly 5KGA, and production of 2KGA was slight, but the production amount was half of that at 30 ° C. The final conversion yield was 50% of the raw material after culturing at 37 ° C. for 96 hours.

<Improvement of 5KGA production at high temperature>
The PQQ-GLDH activity of the wild type and the mutant at 37 ° C. depended on the addition of a cofactor (PQQ and / or Ca ²⁺ ) (FIG. 8a). Various concentrations of CaCl ₂ were added to a medium of Gluconobacter THF55 (gndG :: Km) and cultured at 37 ° C. As a result, 5 KGA accumulation was confirmed after 3 days of culture. Although the effect on growth by addition of CaCl ₂ was not observed, the addition of 5 mM CaCl ₂ produced a high effect on 5KGA production. This was a yield in which 90% of the raw material was converted to 5KGA. Addition of CaCl ₂ at a concentration higher than 5 mM reduced 5KGA production and slightly inhibited growth. From this result, the appropriate concentration of CaCl ₂ for increasing 5KGA production in the culture at 37 ° C. was 5 mM (FIG. 8b).

  The 5KGA fermentation production method using the strain of the present invention eliminates the need for cooling the medium by culturing under high temperature conditions, and can greatly reduce the cost. In addition, since the mutant strain of the genus Gluconobacter of the present invention specifically produces 5KGA, the purification process is facilitated, and therefore the novel strain of the present invention is highly likely to be used industrially. .

It is drawing which shows the enzyme reaction which produces | generates 5KGA and 2KGA from glucose. It is a photograph replaced with drawing which shows the production of 2KGA and 5KGA in the culture medium by the isolate | separated high temperature tolerance Gluconobacter genus bacteria. (1: D-glucose, 2: NaGA, 3: 2 KGA, 4: 5 KGA, 5: medium, 6: THE42, 7: THF55, 8: THG42, 9: THE42, 10: THF55, 11: THG42, where 6 -8 was cultured at 30 ° C, and 9-11 was cultured at 37 ° C.) It is a schematic diagram of the genome fragment obtained from the high temperature resistant Gluconobacter frateuri THF55 strain. It is the photograph replaced with drawing which shows that the variation | mutation in the kanamycin resistance cassette of the high temperature tolerance Gluconobacter genus microbe was confirmed by PCR. (M: λDNA as a marker, 1: THE42, 2: THE42 gndG :: Km, 3: THF55, 4: THF55 gndG :: Km, 5: THG42, 6: THG42 gndG :: Km) It is the photograph replaced with drawing which shows the production of 2KGA and 5KGA by the FAD deficient strain of the high temperature tolerance Gluconobacter genus bacteria. (1: D-glucose, 2: NaGA, 3: 2 KGA, 4: 5 KGA, 5: medium, 6: THE42 gndG :: Km, 7: THF55 gndG :: Km, 8: THG42 gndG :: Km, 9: THE42 gndG :: Km, 10: THF55 gndG :: Km, 11: THG42 gndG :: Km, where 6-8 was cultured at 30 ° C. and 9-11 was cultured at 37 ° C.) It is drawing which shows the activity of the membrane-bound enzyme with respect to D-arabitol and D-glucose of high temperature tolerance Gluconobacter genus bacteria. (A: cultured at 30 ° C., b: cultured at 37 ° C., gray column: activity of D-arabitol cultured with no cofactor added, black column: activity of D-arabitol cultured with 15 μMPQQ and 10 mM CaCl ₂ added, White column: activity of D-glucose cultured without cofactor addition, hatched column: activity of D-glucose cultured with addition of 15 μMPQQ and 10 mM CaCl ₂ ) It is drawing which shows ketogluconic acid production of high temperature tolerance Gluconobacter genus bacteria. (A: cultured at 30 ° C., b: cultured at 37 ° C., gray column: 2 KGA concentration, black column: 5 KGA concentration) In culture at 37 ° C. of temperature resistance Gluconobacter frateurii THF55 strain is a drawing showing the effect of adding CaCl ₂ to Growth and 5KGA producing bacteria. (A: Growth curve (black square: 0 mM, black diamond type 2.5 mM, white triangle: 5 mM, 4-way cross: 7.5 mM, 6-way cross: 10 mM), b: 5 KGA production)

Claims (5)

  A thermotolerant bacterium belonging to the genus Gluconobacter and having the ability to produce 5-keto-D-gluconic acid (5KGA) at 30 to 38 ° C., or a mutant strain thereof, having the ability to produce 5KGA.   A high-temperature-resistant bacterium, wherein the mutant strain according to claim 1 is a mutant strain in which the FAD-gluconate dehydrogenase (FAD-GADH) gene is disrupted.   A mutant strain in which the FAD-GADH gene is disrupted introduces a plasmid into which the kanamycin cassette is inserted into the PCR product obtained by amplifying the DNA fragment having the nucleotide sequence shown in SEQ ID NO: 7, 8, or 9 into the cell. The thermotolerant bacterium according to claim 2, which is a strain constructed by homologous recombination.   Gluconobacter / Frateuri THE42 strain (Accession number NITE-P-655), Gluconobacter / Flateuri THG 42 strain (Accession number NITE-P-657), Gluconobacter / Flateuri THF55 strain (Accession number NITE-P-659) ), Gluconobacter frateuri THE42 gndG :: Km strain (Accession No. NITE-P-656), Gluconobacter frateuri THG42 gndG :: Km strain (Accession No. NITE-P-658), Gluconobacter frateuri THF55 A high-temperature-resistant bacterium having one or more 5KGA producing ability selected from the gndG :: Km strain (Accession No. NITE-P-660).   Culturing the high temperature resistant bacteria belonging to the genus Gluconobacter according to any one of claims 1 to 4 at 30 to 37 ° C in the presence of PQQ and / or Ca ions to produce 5KGA, and collecting 5KGA. A method for producing 5KGA, which is characterized.

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