WO2010070909A1 - High-temperature-resistant bacterium capable of producing 5-ketogluconic acid, and method for producing 5-ketogluconic acid using the high-temperature-resistant bacterium - Google Patents

Abstract

Disclosed is a high-temperature-resistant bacterium which can be applied to the industrial production of 5KGA. Specifically disclosed are: a high-temperature-resistant bacterium which belongs to the genus Gluconobacter and is capable of producing 5KGA at 30 to 38°C, or a mutant strain of the high-temperature-resistant bacterium which is produced by disrupting an FAD-GADH gene in the high-temperature-resistant bacterium; and a method for producing 5KGA, which is characterized by culturing the high-temperature-resistant bacterium at 30 to 37°C in the presence of PQQ and/or a Ca ion to produce 5KGA and collecting 5KGA.

Description

High temperature resistant bacteria having ability to produce 5-ketogluconic acid, and method for producing 5-ketogluconic acid using high temperature resistant bacteria

The present invention relates to a thermotolerant bacterium capable of producing 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 sugar, sugar alcohol, and sugar acid, and accumulate the oxidized product in a medium with high yield. Enzymes involved in these oxidation reactions are dehydrogenases such as D-glucose, D-sorbitol, D-mannitol, glycerol, gluconic acid, ketogluconic acid and the like. All of these dehydrogenases 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 via ubiquinone, generating bioenergy along with the electron transfer reaction. To do.

It is known that the oxidation of D-glucose to keto-D-gluconic acid is catalyzed by an enzyme 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. . The present inventors have reported that the formation of ketogluconic acid found 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, That is, PQQ-glycerol dehydrogenase (PQQ-GLDH: Pyrroloquinoline-quinone-glycerol-dehydrogenase) (FIG. 1). In addition, it has been reported by Shinagawa et al. That FAD-GADH has three subunits (Non-patent Document 2). The three subunits are 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 a wide variety of 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 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 used as a raw material for tartaric acid, xylic acid, vitamin C and important fragrance compounds (Non-patent Document 4). Gray 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 Reichsteind 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). . Also, since 2KGA production is catalyzed by the FAD-GADH gene, a FAD-GADH-deficient mutant of Gluconobacter oxydans 621H (identical to Gluconobacter suboxydans IFO12528) was prepared from D-glucose. 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.

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 a high temperature resistant bacterium applicable to industrial 5KGA production.

In order to screen for strains that can grow 5KGA and grow at a high temperature of 37 ° C. where thermophilic bacteria cannot grow, the present inventors have a total of about 1500 strains, including gluconobacter stocks and isolates from Thailand, etc. As a strain that does not produce a brown compound in a medium using a glucose-gluconic acid medium and develops a color by the resorcinol method, three novel high-temperature-resistant Gluconobacter spp. Wild strain) was isolated. Furthermore, the FAD-GADH gene system of the above-mentioned high temperature resistant wild strain having the ability to produce high 5KGA is used by using a simple method called electroporation without using the complicated method of the three-parent joining method described in Non-Patent Document 5. As a result, a high-temperature-resistant mutant strain capable of specifically producing 5KGA was created, and the present invention was completed.

That is, the present invention provides the following (1) to (6).

(1) A high-temperature tolerant wild strain having the ability to produce 5-keto-D-gluconic acid (5KGA) belonging to the genus Gluconobacter, or a mutant thereof, comprising 20 g glucose, 20 g sodium gluconate, 3 g polypeptone, 3 g yeast 5KGA in which the concentration of 5KGA in the culture supernatant measured with resorcinol reagent is 45 mM or more when cultured in a 2% glucose-gluconic acid medium made up to 1 L with distilled water at 37 ° C. for 72 hours. High-temperature-resistant bacteria that have productivity.

(2) The thermotolerant bacterium according to (1) above, wherein the mutant strain is a mutant strain in which the FAD-gluconate dehydrogenase (FAD-GADH) gene is disrupted.

(3) When a mutant strain in which the FAD-GADH gene is disrupted is cultured in a 2% glucose-gluconic acid medium at 30 ° C. and 37 ° C. for 72 hours, the concentration of 5KGA in the culture supernatant measured using a resorcinol reagent is The thermotolerant bacterium according to (2) above, which is a mutant strain having 5KGA producing ability of 170 mM or more and 90 mM or more, respectively.

(4) 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 NOs: 7, 8, and 9 from a mutant strain in which the FAD-GADH gene is disrupted is introduced into the cell. The high-temperature resistant bacterium according to (2) above, which is a strain constructed by introduction and homologous recombination.

(5) Gluconobacter / Frateuri THE42 strain (Accession number NITE BP-655), Gluconobacter / Flateuri THG42 strain (Accession number NITE BP-657), Gluconobacter Frateuri THF55 strain (Accession number NITE BP-659) , Gluconobacter frateuri THE42 gndG :: Km strain (Accession number NITE BP-656), Gluconobacter frateuri THG42 gndG :: Km strain (Accession number NITE BP-658), Gluconobacter frateuri THF55 gndG: One or more kinds of 5-keto-D-gluconic acid-producing bacteria selected from Km strain (Accession No. NITE BP-660).

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

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. Moreover, since the high temperature resistant mutant strain having 5KGA production ability specifically produces 5KGA, the purification process is easy and industrially advantageous.

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.) FIG. 3 is a schematic diagram of a genome fragment obtained from a 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: Marker λDNA, 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: 2KGA, 4: 5KGA, 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 were cultured at 30 ° C. and 9-11 were cultured at 37 ° C.) 1 is a drawing showing the activity of a membrane-bound enzyme for D-arabitol and D-glucose of a high temperature resistant Gluconobacter genus. (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)

The thermotolerant bacteria of the present invention include 20 g glucose, 20 g sodium gluconate, 3 g polypeptone, 3 g yeast extract in 1 L with distilled water and cultured in a 2% glucose-gluconic acid medium at 37 ° C. for 72 hours. There is no particular limitation as long as it is a high-temperature-resistant wild strain having 5KGA-producing ability belonging to the genus Gluconobacter or a mutant strain thereof having a 5KGA concentration of 45 mM or more in the culture supernatant measured using 2% glucose-gluconic acid When cultured in a medium at 30 ° C. for 48 hours, the 5KGA concentration in the culture supernatant measured using the resorcinol reagent is 50 mM or more, particularly 5 mM or more, particularly high temperature resistant wild strains belonging to the genus Gluconobacter belonging to the genus Gluconobacter The mutant is preferred. As the measurement method using the resorcinol reagent, as described in the examples below, the method described in Methods in Enzymology vol.3 p.248-249 and the Agricultural Chemistry Experiments (Supplement) Volume 2, A method according to the method described in Kyoto University Faculty of Agriculture, Department of Agricultural Chemistry, page 682 can be exemplified. Examples of the genus Gluconobacter include Gluconobacter oxydans, Gluconobacter oxydans, and Gluconobacter frateurii.

The high-temperature resistant wild strain 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 such as a potato agar medium and cultured at 30 ° C. to 37 ° C. for 2 to 3 days. The strain that produced the 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. Preferred examples of the high-temperature-resistant gluconobacter genus strain screened in this manner include Gluconobacter frateuri THE42 strain, Gluconobacter frateuri THG42 strain, and Gluconobacter frateuri THF55 strain.

The above-mentioned wild strains belonging to the genus Gluconobacter were deposited on October 9, 2008 at the Patent Evaluation Microorganism Depositary Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan) Has been. The accession number is Gluconobacter frateuri THE42 strain, the accession number NITE BP-655, Gluconobacter frateuri THG42 strain is the accession number NITE BP-657, Gluconobacter frateuri THF55 strain, the accession number NITE BP- 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. Preferred examples include those that have been specifically modified to produce 5KGA by disrupting the FAD-GADH gene, which is the enzyme to be used. 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. The FAD-GADH gene on the chromosome is disrupted by transforming the fungus with DNA containing a gene that has been modified so that it does not produce a functional enzyme, and causing homologous recombination with a normal gene on the chromosome It can produce by doing. Such gene disruption by homologous recombination gene replacement has already been established as a method using linear DNA or plasmid. The production of the FAD-GADH gene disrupted strain of the high-temperature-resistant Gluconobacter bacterium according to the present invention can be performed, for example, by the following operation method and procedure.

1) Extract DNA from high temperature resistant Gluconobacter spp. Genomic DNA extraction from high-temperature-resistant Gluconobacter spp. (Sambrook, brookJ., Et al, 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor , NY).

2) Cloning the target FAD-GADH gene. Cloning of the FAD-GADH gene can be performed by PCR using primers designed from the region maintained in the dehydrogenase subunit in the gene of the 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 on the basis of the FAD-GADH gene sequence reported in the Gluconobacter oxydans 621H genome. 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. Using ABI PRISM 310 (manufactured by PE Biosystems), the entire sequence of the cloned gene is software such as GENETYX-MAC (Software Development, Tokyo, Japan) and Clone Manager (Scientific and Educational Software, Cary, NC) To obtain sequence data. For homology search and alignment analysis, BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) and CLUSTAL W (www.ebi.ac.uk/clustalw) are used, 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. A preferred example is gndG that encodes a subunit that contains. These DNA fragments can be amplified and used as a plasmid for FAD-GADH gene disruption.

3) Prepare a FAD-GADH gene disruption plasmid based on the sequence information of the cloned gene. A plasmid for disrupting the FAD-GADH gene was obtained 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 disruption strain is prepared by introducing a plasmid. The FAD-GADH gene disruption strain was prepared by subjecting the gene disruption plasmid 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 highly efficient gene transfer methods such as transconjugation, and FAD-GADH gene is disrupted (or inactivated) by homologous recombination to chromosomes. Can be performed. Confirmation of the disruption of the FAD-GADH gene is performed by applying a strain having a drug resistance gene fragment inserted into a chromosome onto a plate medium containing an appropriate concentration of the drug, and transforming the strain into a high temperature resistant Gluconobacter genus. This can be done by selecting bacteria and confirming that the selected bacterial strain has lost enzyme activity for 2KGA production.

The mutant strain disrupted in the FAD-GADH gene prepared as described above was cultured using a resorcinol reagent when cultured in a 2% glucose-gluconic acid medium at 30 ° C. and 37 ° C. for 72 hours. Suitable examples include mutants having 5KGA production ability, in which the concentration of 5KGA in the supernatant is 150 mM or more, preferably 170 mM or more, and 80 mM or more, preferably 90 mM or more. In addition, the transformed high-temperature-resistant Gluconobacter genus of the present invention can be prepared by using an appropriate medium such as potato agar medium (20 g glycerol, 5 g glucose, 10 g yeast extract, 10 g polypeptone, 2 g agar, 100 ml potato extract with distilled water. 1L) and 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.

The following are examples of mutant strains of the transformed high temperature resistant Gluconobacter genus of the present invention. The mutant strain of Gluconobacter frateuri THE42 strain (Accession number NITE BP-655) is Gluconobacter frateuri THE42 gndG :: Km strain, the accession number NITE BP-656, and Gluconobacter frateuri THG 42 The mutant strain of the strain (Accession No. NITE BP-657) is Gluconobacter Frateuri THG42 gndG :: Km strain, the accession No. NITE BP-658, and the Gluconobacter Frateuri THF 55 strain (Accession No. NITE BP-) 659) is Gluconobacter frateuri THF55 gndG :: Km, and has the accession number NITE BP-660, respectively, and the National Institute of Technology and Evaluation of the National Institute of Technology and Evaluation (1000 Japan) Has been deposited on October 9, 2008 in the prefecture Kisarazu Kazusa legs bowed in 2-5-8).

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 which 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, magnesium sulfate, or the like 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, it is preferable to add a cofactor to the medium for the enzyme involved in 5KGA production. 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 added to the medium for the enzyme activity involved in the production of 5KGA 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 to 100 μM, preferably 1 to 10 μM. PQQ can be used in combination with the magnesium ion, calcium ion, ammonium ion, sodium ion, potassium ion and the like.

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 the culture is preferably in the range of about 5 to 7, and the pH during the culture can be adjusted by adding an acid or an alkali. By culturing for 1 to 7 days, preferably 2 to 5 days, 5KGA can be obtained. Production of 5KGA is seen in the first growth stage, but the highest production is seen in the stable growth period.

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

<Screening of high temperature resistant 5KGA production strain of Gluconobacter>
1. Wild strain screening All Gluconobacter strains used in the screening experiments were isolated from various Thai materials by the method of Moonmangmee, D. et al. (Bioscience, Biotechnology and Biochemistry 64: 2306-2315, 2000). It was stored in an agar medium (20 g glycerol, 5 g glucose, 10 g yeast extract, 10 g polypeptone, 2 g agar, 100 ml of potato extract made up to 1 L with distilled water). 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) and 30 ° C. The strains that were cultured for 48 hours at 37 ° C. or 60 hours at 37 ° C. and produced brown compounds in the medium were excluded as 2,5-diketogluconic acid producing strains. Thereafter, 200 μl of the medium and 1 ml of Resorcinol reagent (49% of 0.5% Resorcinol, 168 ml of concentrated hydrochloric acid, 273 ml of distilled water) 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 production 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 a 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 enzymatically quantified with 2KGA reductase and 5KGA reductase, respectively, according to the method of Saichana. 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). Plates were dried and sprayed with a coloring 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 IFO 12528 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 the FAD-gluconate dehydrogenase gene from thermotolerant Gluconobacter fungus The 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 represented by Elvinia cypripedi (Journal of Bacteriology 179: 6566-6572, 1997), Gluconobacter dioxyacetonicus IFO 3271 (Microbiology 73: 6551-6556, 2007), Gluconobacter oxydans 621H (Nature Biotechnology) 23: 195-200, 2005) from the region conserved in the dehydrogenase subunit in the GADH gene. PCR was carried out using this primer pair 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 with QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) or MagExtracter DNA fragment purification 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. Using No. 4), PCR was performed by adding ₂ mM MgCl ₂ and 0.5% DMSO to TaKaRa LA Taq ™ (manufactured by 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 F-igL-1 (SEQ ID NO: 3) and R-igL-2 of primers using a template obtained by recirculating a SacII or EcoRI digest of genomic DNA. Amplified with (SEQ ID NO: 4).
The cytochrome C subunit gene was obtained from an in vitro cloning method.

Sequences were identified by sequencing 6 ORF fragments containing 3 ORFs (gndF, gndG, gndH) predicted from the FAD-GADH gene obtained from each strain and 3 ORFs encoding proteins with unknown functions. The sequencing was performed using ABI PRISM 310 (PE Biosystems). Sequence data was analyzed using GENETYX-MAC (Software Development Corporation) and Clone Manager (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 has already been registered as Accession No.４７AB478873 in DNA Data Bank of Japan (DDBJ).

The determined amino acid sequence of GndF was reported by Berks, B. C. et al. (Molecular Microbiology 35: 260-274, 2000), and there was a tat-dependent special signal sequence containing two arginine residues. I found out that GndF is also a small subunit of FAD-GADH. To GndS of Gluconobacter dioxyacetonicus IFO3271 revealed in a report by Toyama.H et al. (Applied バ Environmental Microbiology 73: 6551-6556, 2007) % Identity.

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 in the glucose-methanol-choline (GMC) oxidoreductase family, a protein family that binds FAD as a cofactor. Further, it showed 61% identity with GndL of FAD-GADH dehydrogenase subunit of Gluconobacter dioxyacetonicus IFO3271 found by Toyama.

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 logarithmic growth phase according to Marmur's method (J. Mol. Biol. 3: 208-218, 1961). Isolated from Novacter. A plasmid for disrupting 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 obtained. 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, the 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 resistance to ampicillin were selected. The gene disruption was confirmed by PCR using a primer that covers and amplifies 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 for D-arabitol and glucose in wild and mutant strains was prepared from cells grown at 30 ° C. and 37 ° C. by membrane fractionation. 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 ferricyanide. Of 0.1 ml of 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. 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 fungus grows at high temperature.

<Production of 5KGA by FAD-GADH-deficient mutant>
Wild 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 b). 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 wild-type and mutant PQQ-GLDH activities at 37 ° C. depend on the addition of cofactors (PQQ and / or Ca ²⁺ ). Accordingly, various concentrations of CaCl ₂ were added to a medium of Gluconobacter THF55 (gndG :: Km) and cultured at 37 ° C. (FIG. 8a). As a result, accumulation of 5KGA 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).

In the 5KGA fermentation production method using the strain of the present invention, it is not necessary to cool the medium by culturing under high temperature conditions, and the cost can be greatly reduced. 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. .

Claims (6)

1. A high-temperature-resistant wild strain having the ability to produce 5-keto-D-gluconic acid (5KGA) belonging to the genus Gluconobacter, or a mutant thereof, and distilled 20 g glucose, 20 g sodium gluconate, 3 g polypeptone, 3 g yeast extract 5KGA production ability characterized by the fact that when cultured in a 2% glucose-gluconic acid medium made up to 1 L with water at 37 ° C. for 72 hours, the 5KGA concentration in the culture supernatant measured using resorcinol reagent is 45 mM or more. High temperature resistant bacteria.
2. 2. The thermotolerant bacterium according to claim 1, wherein the mutant strain is a mutant strain in which the FAD-gluconate dehydrogenase (FAD-GADH) gene is disrupted.
3. When a mutant strain in which the FAD-GADH gene was disrupted was cultured in a 2% glucose-gluconic acid medium at 30 ° C. and 37 ° C. for 72 hours, the 5KGA concentration in the culture supernatant measured using a resorcinol reagent was 170 mM or more. The high-temperature-resistant bacterium according to claim 2, which is a mutant having 5 KGA producing ability of 90 mM or more.
4. 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 NOs: 7, 8, and 9 into the cell. The thermotolerant bacterium according to claim 2, which is a strain constructed by homologous recombination.
5. Gluconobacter frateuri THE42 strain (Accession number NITE BP-655), Gluconobacter frateuri THG42 strain (Accession number NITE BP-657), Gluconobacter frateuri THF55 strain (Accession number NITE BP-659), Glucono Bacter Frateuri THE42 gndG :: Km strain (Accession number NITE BP-656), Gluconobacter Frateuri THG42 gndG :: Km strain (Accession number NITE BP-658), Gluconobacter Frateuri THF55 gndG: Km strain One or more kinds of 5-keto-D-gluconic acid-producing bacteria selected from the trust number NITE BP-660).
6. The high-temperature resistant bacterium according to any one of claims 1 to 5 is cultured in the presence of pyrroloquinoline quinone and / or Ca ions at 30 to 37 ° C to produce 5KGA, and 5KGA is collected. 5KGA manufacturing method.
   

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