KR100542563B1 - Microorganism producing riboflavin and method for producing riboflavin using thereof - Google Patents

Abstract

The present invention relates to a Bacillus subtilis mutant strain that produces riboflavin and is resistant to the antibiotic trimethoprim, and a method for producing riboflavin using the microorganism. Bacillus subtilis mutant CJKB0003 according to the present invention is resistant to trimethoprim, an antibiotic that inhibits dihydrofolate reductase, which biosynthesizes tetrahydrofolate during microbial metabolism. Therefore, riboflavin is characterized by producing high concentration and high yield. Therefore, the present invention has the effect of culturing the microorganism and obtaining a large amount of riboflavin from the culture.

Riboflavin, Bacillus subtilis, Trimethoprim, Antibiotic Resistance

Description

Microorganisms producing riboflavin and method for producing riboflavin using same

The present invention relates to a microorganism producing riboflavin and a method for producing riboflavin using the same. More specifically, the present invention relates to a Bacillus subtilis mutant CJKB0003 characterized by producing riboflavin and being resistant to the antibiotic trimethoprim, and a method of producing riboflavin using the microorganism.

Riboflavin (vitamin B2) is a water-soluble vitamin that is biosynthesized by various species of microorganisms and all plants. However, there is a property that is not biosynthetic in vertebrates including humans. Riboflavin is a precursor of flavin adenine dinucleotide (FAD) and flamin mononucleotide (FMN), a coenzyme required for the oxidation-reduction of all organic cell bodies, and is an essential nutrient for animals including humans. Deficiency of riboflavin can cause inflammation of the oral and pharyngeal mucosa, skin inflammation and other skin injuries, conjunctivitis, vision loss, growth loss and weight loss. Thus, riboflavin has been used as a vitamin preparation for the prevention or treatment of diseases associated with vitamin deficiency as described above and as a feed additive for livestock growth. In particular, concentrated riboflavin has been used as a feed in itself. Currently, the production of riboflavin is 6000 tonnes annually, about 75% of which is used as feed additive, and the rest is used for food and medicine.

Currently, as a method of producing riboflavin, a chemical synthesis method and a fermentation method using microorganisms are used in combination. Chemical synthesis is a method of producing high purity riboflavin through a multi-step process, usually using a precursor such as D-ribose. The chemical synthesis method has a disadvantage that the production cost is expensive because the starting material is expensive, a fermentation method using a microorganism has been developed. Fermentation method using microorganism is to isolate the riboflavin-producing microorganism from nature or to incubate the microorganism which induced the mutation by genetic engineering method or chemical-physical method under appropriate conditions so that riboflavin is overproduced and then separate riboflavin from the culture. It is a way.

A microorganism producing the riboflavin is Mai in process as Saccharomyces (Saccharomyces sp.) And the Candida genus (Candida sp.) Yeast, genus Clostridium (Clostridium sp.) Belonging to, Bacillus (Bacillus sp.) And Corey Yes tumefaciens in (Corynebacterium sp.) and bacteria belonging to the array: Mote when help in (Eremothecium ashbyii sp.) and there are known such as fungi belonging to the genus Ashton vias (Ashbya sp.).

U.S. Patent No. 5,231,007 discloses a method for preparing riboflavin using the yeast Candida famata , and the literature describes Bacillus subtilis and Corynebacte engineered to overexpress riboflavin biosynthesis-related enzyme genes. Examples of producing riboflavin of 4.5 g / L and 17.4 g / L, respectively, have been reported using Corynebacterium ammoniagenes (Perkins et al., J. Ind. Microbiol. Biotechnol. , 22: 8). -` 8, 1999). European Patent No. 0821063 discloses a method for producing riboflavin using recombinant Bacillus subtilis, and US Pat. No. 5,837,528 introduces a vector having rib operon to Bacillus subtilis using recombinant technology to improve the productivity of riboflavin. Increased recombinant strains are disclosed. In addition, U.S. Patent No. 5,334,510 discloses a riboflavin production method using a mutant strain of Bacillus subtilis that attenuates 5'-GMP resolution to improve riboflavin productivity. In the industrial production of riboflavin, it Ascomycetes the array: Mote when a strain is helpful Ashton ratio (Eremothecium ashbyii) and Ashton via blood examination (Ashbya gossypii) are the most common. The mutant strains of the Aspergillus fungus were cultured in a nutrient medium containing molasses or vegetable oil as a main carbon source to produce 15 g of riboflavin per liter of fermentation broth. Regarding the production of riboflavin using the Ashbya gossypii, it has been disclosed in International Patent Publication No. 9526406.

However, despite the above research achievements, industrial mass production of riboflavin is still required, and thus, there is a need to further develop microorganisms having improved production capacity of riboflavin.

Meanwhile, in the fermentation method using microorganisms, strains having resistance to various antibiotics have been developed to obtain culture products at high concentration and high yield. The result of a study of improving productivity by introducing into a lysine producing strain having resistance to the antibiotic monensin (Korean Patent Laid-Open Publication 2002-0058956), cymidine using a strain resistant to the antibiotic trimethoprim used in the present invention (thymidine) The productivity improvement of various fermentation products by antibiotic-resistant strains, such as the results of studies that improve the productivity (Korean Patent Publication No. 2002-0046602) is known.

The present inventors, while studying the microorganisms with improved production capacity of riboflavin, by giving resistance to the antibiotic trimethoprim to the Bacillus subtilis, the biosynthesis of purin, a precursor of riboflavin is enhanced during the metabolic process of microorganisms compared to the parent strain The present invention was completed by selecting mutant strains that produce remarkably high concentrations and high yields of riboflavin and using them to produce high concentrations of riboflavin.

It is therefore an object of the present invention to provide a Bacillus subtilis mutant CJKB0003 that produces riboflavin.

Another object of the present invention is to provide a method for producing riboflavin using the microorganism.

In order to achieve the above object, the present invention provides a Bacillus subtilis mutant CJKB0003 (KCCM-10526) characterized by producing riboflavin and having resistance to trimethoprim.

The present invention also provides a method for producing riboflavin using the Bacillus subtilis mutant CJKB0003.

Hereinafter, the present invention will be described in detail.

Bacillus subtilis mutant CJKB0003 according to the present invention is a mutant of Bacillus subtilis CJKB0001 producing riboflavin and is an antibiotic that inhibits enzymes involved in the biosynthesis of tetrahydrofolate during microbial metabolism. As a result of the enhanced resistance to trimethoprim, the riboflavin biosynthetic pathway is consequently strengthened, thus producing riboflavin at a high concentration and high yield in comparison to the parent strain Bacillus subtilis CJKB0001. Trimethoprim is 5 - ((3,4,5-Trimethoxyphenyl) methyl) with diamino-pyrimidine (diaminopyrimidine) having the chemical name of -2,4-pyrimidinediamine action both on Gram-positive and Gram-negative bacteria, especially E.coli , Antibiotics used for the treatment of urethral and respiratory infections caused by Proteus mirabilis and Klebsiella pneumoniae . The antimicrobial mechanism of trimethoprim is known to be due to inhibition of bacterial folate biosynthesis. Trimethoprim binds more than 50000 times more strongly to bacterial dehydrofolate reductase than acts on the body's dihydrofolate reductase. Trimethoprim in this case inhibits the biosynthesis of tetrahydrofolate from dihydrofolate by binding to dihydrofolate reductase and inhibiting enzyme action. Tetrahydrofolate transfers one-carbon fragments between molecules and plays an important role in the biosynthesis of thymidine, purines and amino acids. By this mechanism, trimethoprim selectively inhibits the nucleic acid and protein biosynthesis of bacteria to exhibit an antimicrobial effect.

It can be inferred from this series of mechanisms that the biosynthesis of tetrahydrofolate will be enhanced for strains with enhanced resistance to trimethoprim. Tetrahydrofolate, on the other hand, acts as a coenzyme in the reaction of delivering one carbon unit in purine biosynthesis. In conclusion, the enhanced biosynthesis of tetrahydrofolate by the introduction of resistance to trimethoprim is thought to increase the productivity of riboflavin by enhancing the biosynthesis of purine, a precursor of riboflavin.

Therefore, the present inventors have treated the mutagen to give the Bacillus subtilis CJKB0001 resistance to trimethoprim, inducing mutations, and then having a resistance to trimethoprim that can grow in a medium containing trimethoprim. Were selected, and among them, the strains with the best riboflavin production ability were selected.

Bacillus subtilis CJKB0001 ( Bacillus subtilis CJKB0001) used as the parent strain has been filed in Korean Patent No. 10-2002-076868. As a method of inducing mutations in the parent strain, physical or chemical methods known in the art may be used. For example, X-rays or ultraviolet rays may be used as physical methods, and N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and diethyl may be used as chemical methods. Chemical modifiers such as sulfate and ethylamine may be used.

The present inventors cultivated the strains induced by the mutation in the medium containing the antibiotic trimethoprim by concentration as described above to select the mutants capable of growing in the presence of a high concentration of trimethoprim, among which the best riboflavin production ability One week of variation was selected. It was named Bacillus subtilis mutant CJKB0003 and was deposited with the Korean Culture Center of Microorganisms on November 17, 2003 and was given accession number KCCM-10526.

Bacillus subtilis mutant CJKB0003 (KCCM-10526) according to the present invention has the characteristics that can be grown even in a medium containing up to 130㎛ / l trimethoprim. On the contrary, Bacillus subtilis CJKB0001, which is used as a parent strain, is capable of growing in a medium containing less than 100 µg / l of trimethoprim and has a characteristic of not growing at higher concentrations.

Thus, the Bacillus subtilis mutant CJKB0003 of the present invention is endowed with resistance to trimethoprim, thereby alleviating the inhibition by trimethoprim of dihydrofolate reductase during metabolic process of the strain and propelling riboflavin biosynthesis. As a result, the biosynthesis of purines, which is a substance, is strengthened, and as a result, the riboflavin production ability is remarkably improved compared to the parental strain Bacillus subtilis CJKB0001.

The present inventors cultured Bacillus subtilis mutant CJKB0003 according to the present invention in a flask, and confirmed that it accumulates 9.1 g / l of riboflavin in a medium having a concentration of about 13.8% higher than that of the parent strain Bacillus subtilis CJKB0001. It was. In addition, the Bacillus subtilis mutant strain CJKB0003 according to the present invention was cultured in 51 fermentor, and it was confirmed that 29.6 g / l of riboflavin accumulated in the medium was about 10.4% higher than the parent strain.

Therefore, Bacillus subtilis mutant CJKB0003 (KCCM-10526) according to the present invention is a strain with improved resistance to antibiotics trimethoprim compared to the parent strain is remarkably improved riboflavin production capacity by strengthening the purine biosynthesis.

The present invention also provides a method of producing riboflavin from the culture by culturing the Bacillus subtilis mutant CJKB0003 of the present invention under suitable conditions.

That is, the Bacillus subtilis mutant CJKB0003 (KCCM-10526) is inoculated in a conventional medium containing a suitable carbon source, nitrogen source, amino acids and vitamins, and cultured by adjusting the temperature and pH under aerobic conditions. The carbon source may be glucose, molasses, lactose, sugar, sucrose, maltose, dextrin, starch, mannitol, sorbitol, and sorbitol. Glycerol may be used. Preferably, glucose and molasses are used. As the nitrogen source, various inorganic nitrogen sources such as ammonia, ammonium chloride, ammonium sulfate, peptone, NZ-amine, meat extract, yeast Organic nitrogen sources such as extracts, corn steep liquors, casein hydrolysates, fish or degradation products thereof, skim soy cakes or degradation products thereof can be used. Preferably, yeast extract and corn steep liquor are used. The inorganic compound may include potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, and calcium carbonate. carbonate) and the like, and vitamins and nutrient bases may be additionally added as necessary. The culturing is carried out at a temperature of 30 to 45 ° C., preferably 37 to 40 ° C., under aerobic conditions, for example, by shaking culture or aeration stirred culture. The pH of the medium is preferably maintained near the neutral during the culturing, and the culture period is performed for 2 to 6 days.

In one embodiment according to the present invention inoculated Bacillus subtilis mutant CJKB0003 of the present invention to the seed medium and incubated for 20 hours at 37 ℃, 800rpm while supplying air at 1vvm, inoculated the seed culture solution in the fermentation medium by air Was fed at 1 vmv and shaken at 40 ° C., 800 rpm, pH 7.0 for 60-70 hours, while supplying an additional medium containing glucose so that the residual sugar concentration in the culture was 0.5-1%, and the total sugar content in the culture was 27%. Riboflavin was obtained at a concentration of about 10.4% improved compared to the parent strain by culturing by addition.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1: Selection of Bacillus subtilis mutant CJKB0003 according to the present invention

Bacillus subtilis mutant CJKB0003 of the present invention is treated with a mutagen to the parent strain Bacillus subtilis CJKB0001 to induce mutations and then cultured in a medium containing trimethoprim to obtain a colony capable of growing riboflavin This was done by selecting the best mutant strains.

Bacillus subtilis CJKB0001 used as a parent strain is a strain filed in Korean Patent No. 10-2002-076868. The Bacillus subtilis CJKB0001 of phosphate buffer and suspended in a concentration of 10 ⁷ ~ 10 ⁸ cells / ml in (phosphate buffer, pH7.0) or citrate buffer (citrate buffer, pH5.5). To the suspension, a mutagenic agent, N-methyl-N'-nitro-N-nitrosoguanidine, was added so that the final concentration was 10-50 µg / ml. This was treated for 30 to 60 minutes at room temperature or 30 ° C. to induce mutations. After washing three times with 0.85% saline and diluting appropriately, trimethoprim was added to a minimum medium containing 1.8% agar in different concentrations to prepare a medium, and then smeared on the medium containing trimethoprim. After incubation at 37 ℃ for 5 days to obtain a colony (colony). At this time, the concentration of trimethoprim was adjusted to 0 mg / l to 140 mg / l. Colonies grown in minimal medium containing various concentrations of trimethoprim were inoculated in nutrient medium and incubated for 24 hours at 37 ° C. Strains with the highest yield of riboflavin were selected. The amount of riboflavin produced was analyzed using HPLC.

The analysis method of riboflavin using HPLC is as follows.

Device: Water 510

Column size: inner diameter 4.6mm, length 250mm

Filler: Chromasil C18 5um

Mobile phase: A. 5 mM Sodium hexanesulfonate

+ 20 mM H ₃ PO ₄

        B. Acetonitrile

        A: B = 89: 11

Flow rate: 1ml / min

Detector: TSP UV2000 (UV 260 nm)

Sample injection volume: 15ul

Sample solvent: distilled water

Each medium composition used in the experiment is shown in Table 1.

Medium composition for selection of Bacillus subtilis mutant CJKB0003 according to the invention badge Furtherance Nutritional Agar Badge Trypsose 10g / l, Beef Extract 3g / l, Sodium Chloride 5g / l, Agar 15g / l Minimal badge Glucose 2.5g / l, Potassium phosphate 7g / l, Dipotassium phosphate 3g / l, Sodium citrate 0.5g / l, Magnesium sulfate heptahydrate 0.75g / l, Yeast extract 0.5g / l, Casamino acid 0.15g / l, tryptophan 20mg / l Trimethoprim medium Medium containing trimethoprim 40-140 ㎍ / l in minimal medium Fermentation medium Glucose 100g / l, Dry yeast 20g / l, Magnesium sulfate heptahydrate 0.5g / l, Potassium phosphate 1.5g / l, Dipotassium phosphate 3.5g / l, Urea 6g / l, Erythromycin 10mg / l, Chloramphenicol 10 mg / l, pH 7.2 ~ 7.4

 As a result of the culture, one strain of the best strain of riboflavin was selected among the strains grown in a medium containing 130 µg / l of trimethoprim and named as Bacillus subtilis strain CJKB0003. The selected Bacillus subtilis mutant strain CJKB0003 was deposited with the Korean Culture Center of Microorganisms on November 17, 2003 and was assigned accession number KCCM-10526.

Example 2: Comparison of resistance to Trimethoprim of Bacillus subtilis mutant CJKB0003 according to the present invention and Bacillus subtilis CJKB0001 used as parent strain

Bacillus subtilis mutant CJKB0003 (KCCM-10526) selected from Example 1 and Bacillus subtilis CJKB0001, which were used as a parent strain, were cultured in a medium to which trimethoprim was added to compare the resistance to trimethoprim. . That is, each strain was inoculated into the trimethoprim medium as shown in Table 1 of Example 1 and incubated at 37 ° C. for 5 days.

As a result, as shown in Table 2, the Bacillus subtilis CJKB0001 strain used as the parent strain could grow in a medium to which trimethoprim was added, but not at a concentration higher than that. In contrast, the Bacillus subtilis mutant CJKB0003 according to the present invention was able to grow in a medium containing 130 µg / l of trimethoprim.

Comparison of Tolerance to Trimethoprim of Bacillus subtilis Mutant CJKB0003 and Bacillus subtilis CJKB0001 Strain / Tiaproline Concentration (㎍ / l) 0 40 60 100 120 130 140 Bacillus subtilis CJKB0001 +++ ++ + + - - - Bacillus subtilis CJKB0003 (KCCM-10526) +++ +++ +++ ++ ++ + -

+: Grow,-: fail to grow

Example 3: Flask fermentation titer of Bacillus subtilis mutant CJKB0003 according to the present invention

The flask fermentation titer of Bacillus subtilis CJKB0003 (KCCM-10526) according to the present invention was investigated in comparison with the fermentation titer of the parent strain Bacillus subtilis CJKB0001. Mutant strains and parent strains according to the present invention were inoculated in the seed medium, and then cultured, followed by fermentation of the seed culture solution in a flask fermentation medium. The seed medium was prepared by dispensing 5 ml in a test tube having a diameter of 18 mm and then autoclaving it at 121 ° C. for 15 minutes. Mutant strains and mother strains according to the present invention were inoculated and shaken for 20 hours at 200 rpm and 37 ° C. When the culture was completed, 1 ml of the seed culture solution was inoculated into the flask fermentation medium and shaken at 200 rpm and 37 ° C. for 90 to 96 hours. The flask fermentation medium was prepared by autoclaving the medium and the starlet medium in the same manner as the seed medium, and then dispensing 15 ml and 5 ml, respectively, in a 250 ml flask previously autoclaved. Upon completion of the culture, the amount of riboflavin accumulated in the medium was analyzed. Riboflavin concentrations were analyzed by HPLC, in which case the assay conditions were as described above.

The composition of the seed medium and the fermentation medium used in the experiment is shown in Table 3.

Medium composition for flask fermentation badge Furtherance Species Yeast extract 5g / l, tryptone 10g / l, sodium chloride 10g / l, no pH adjustment Fermentation medium Main medium: glucose 100g / l, dry yeast 20g / l, magnesium sulfate heptahydrate 0.5g / l Starch medium: 1.5g / l potassium phosphate, 3.5g / l potassium phosphate, urea 6g / l, erythromycin 10mg / l, Chloramphenicol 10mg / l, pH 7.2 ~ 7.4

As a result, the riboflavin accumulation in the medium of the parent strain Bacillus subtilis CJKB0001 was 8.0g / l, and the accumulation of riboflavin in the medium of the Bacillus subtilis mutant strain CJKB0003 was 13.8% higher than that of the parent strain 9.1. g / l.

Example 4: Fermentation titer in 51 fermenter of Bacillus subtilis mutant CJKB0003 according to the present invention

 The fermentation titer of 51 Bacillus subtilis CJKB0003 (KCCM-10526) according to the present invention in the fermenter was investigated in comparison with the fermentation titer of the parent strain Bacillus subtilis CJKB0001. Mutant strains and parent strains according to the present invention were inoculated in the seed medium, followed by seed culture, followed by seed culture inoculation into the fermentation medium to perform fed-batch fermentation. First, seed medium was dispensed by 11 into a 2.5L experimental fermenter and then prepared by sterilizing for 10 minutes at 121 ℃. After cooling, the Bacillus subtilis mutant strain CJKB0003 and the parent strain of the present invention were respectively inoculated in saline and inoculated by 10 ml, and then inoculated with sterilized air at 1vvm and incubated for 20 hours while stirring at 800 rpm at 37 ° C. The pH was not adjusted during incubation. When the seed culture was completed, the seed culture solution was inoculated into the fermentation medium to perform fed-batch fermentation. The fermentation medium was prepared by dispensing 1.41 in a 51-volume experimental fermenter and then autoclaving for 20 minutes at 121 ℃. After cooling it, the seed culture solution was inoculated 200 ml each and incubated at 800rpm and 40 ° C while supplying the sterilized air at 1vvm. At this time, while controlling the residual sugar concentration in culture to 0.5 ~ 1% by supplying an additional medium to the total sugar concentration added to the fermentation broth was 27%. The pH of the culture was incubated for 60 ~ 70 hours, adjusting to 7.0 using ammonia water. After incubation was completed, the accumulation of riboflavin in the medium was analyzed by HPLC. Riboflavin analysis by HPLC is as described above.

The composition of the medium used in the present invention is as shown in Table 4.

51 Composition of medium for fermenter culture badge Furtherance Species Molasses (50% sugar) 30 g / l, corn steep liquor 15 g / l, magnesium sulfate heptahydrate 0.5 g / l, ammonium sulfate 5 g / l, urea 3 g / l, erythromycin 10 mg / l, chloramphenicol 10 mg / l, pH 7.4 Fermentation medium Main medium: 20 g / l dry yeast, 5 g / l corn steep liquor, 2 g / l ammonium sulfate, 0.5 g / l magnesium sulfate heptahydrate, 17.5 g / l potassium phosphate, 7.5 g / l potassium phosphate, erythro Mycin 5mg / l, Chloramphenicol 10mg / l, pH 7.2 ~ 7.4 Additional Medium: Glucose 270g / l, Dry Yeast 26.7g / l, Corn Soak 26.7g / l

As a result, the accumulation of riboflavin in the medium was 26.8 g / l of the parent strain Bacillus subtilis CJKB0001, and in the case of the mutant strain CJKB0003 according to the present invention, it was about 29.6 g / l improved by about 10.4% compared to the parent strain.

As described in the above examples, Bacillus subtilis mutant CJKB0003 according to the present invention is a trimethoprim enzyme that inhibits tetrahydrofolate biosynthesis, a coenzyme involved in purine biosynthesis, a precursor of riboflavin, during microbial metabolism. Because of its resistance to purine biosynthesis, it is possible to produce riboflavin at high concentration and high yield significantly compared to the parent strain Bacillus subtilis CJKB0001. Therefore, the present invention has the effect of culturing the microorganism and obtaining a large amount of riboflavin from the culture.

Claims (3)

Bacillus subtilis CJKB0003 KCCM-10526, which produces riboflavin and is resistant to trimethoprim. delete A method of producing a riboflavin from the culture by culturing the microorganism according to claim 1.