KR100542565B1 - Microorganism producing riboflavin and method for producing riboflavin using thereof - Google Patents

Abstract

The present invention relates to a Bacillus subtilis mutant CJKB0011 producing riboflavin and a method for producing riboflavin using the microorganism. Bacillus subtilis mutant CJKB0011 according to the present invention has a very low concentration of acetoin, a by-product of fermentation, compared to the parent strain, thereby producing riboflavin at a 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, Acetoin

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, as a mutant of Bacillus subtilis CJKB0001 (Korean Patent Application No. 10-2002-076868) that produces riboflavin, acetoin (acetoin, 3-hydroxy-2-butanone), which is a major fermentation byproduct, is used. The present invention relates to Bacillus subtilis mutant CJKB0011 and a method for producing riboflavin using the microorganisms, which are produced at very low levels and significantly improved riboflavin production capacity compared to the parent strain.

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 for this purpose, there is a need to further develop microorganisms with improved production capacity of riboflavin.

 Therefore, the present inventors randomly induce mutations for the purpose of lowering fermentation by-products of Bacillus subtilis while researching microorganisms having improved riboflavin production capacity and using Bacillus subtilis using a high-throughput-screening (HTS) system. Among the major fermentation by-products, mutants with very low acetoin production were selected. As a result, compared to the parent strain Bacillus subtilis CJKB0001 was successfully selected for mutant strains to produce a high concentration and high yield of riboflavin, using this to complete the present invention by producing a high concentration of riboflavin.

It is therefore an object of the present invention to provide a Bacillus subtilis mutant CJKB0011 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 CJKB0011 (KCCM-10525) for producing riboflavin.

In addition, the present invention provides a method for producing riboflavin using the Bacillus subtilis mutant CJKB0011.

Hereinafter, the present invention will be described in detail.

Bacillus subtilis mutant CJKB0011 according to the present invention is a mutant of Bacillus subtilis CJKB0001 ( Bacillus subtilis CJKB0001) that produces riboflavin and generates acetoin, a fermentation byproduct when fermenting glucose as a carbon source, to a very low level. It is a novel strain with the characteristic of producing high concentration and high yield.

A major axis of research to develop industrially available microorganisms is to increase the availability of carbon sources by inhibiting or eliminating the ability to produce their primary fermentation by-products, thereby increasing the concentration of the target product. Generally fermentation using Bacillus subtilis 2,3-butanediol, acetic acid, ethanol, succinic acid and acetoin Produced as a primary fermentation byproduct. In addition, Bacillus subtilis CJKB0001, the parent strain of the present invention, has the property of producing the most acetoin as a main fermentation by-product when fermenting glucose as a carbon source. Acetoin biosynthesis from pyruvate of Bacillus subtilis has been shown to consist of two-step reactions with acetolactate synthase and acetolactate decarboxylase (Renna et al. al., Journal of Bacteriology , 3863-3875, 1993). In addition, when the acetoin biosynthesis gene of Bacillus subtilis was eliminated and the acetoin-producing ability was eliminated, it was reported that the growth of Bacillus subtilis under anaerobic conditions was not significantly affected (Ramos et al., Journal of Bacteriology, 3072-3080, 2000).

In view of the above, the Bacillus subtilis mutant CJKB0011 according to the present invention induces mutations in the Bacillus subtilis producing riboflavin, thereby greatly improving the production capacity of riboflavin by significantly lowering the ability to produce acetoin, the main fermentation byproduct. Strains.

Bacillus subtilis CJKB0001 used as the parent strain is 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 cultured the strains induced by mutations in acetoine-producing selection medium and performed Voges-Proskauer test to select low-producing acetoin strains, and among them, one strain with the best riboflavin production capacity was selected. . It was named Bacillus subtilis mutant CJKB0011 and was deposited with the Korean Culture Center of Microorganisms on November 17, 2003 and received accession number KCCM-10525.

Bacillus subtilis mutant CJKB0011 (KCCM-10525) according to the present invention is characterized by producing 1.1 g / l of acetoin as a fermentation by-product during fed-batch culture using glucose (270 g / l) as a carbon source. On the other hand, Bacillus subtilis CJKB0001 used as a parent strain has the property of producing 4.2 g / l of acetoin under the same conditions. That is, Bacillus subtilis mutant CJKB0011 according to the present invention has a feature that the acetoin generating ability is reduced to about 26.2% of the parent strain.

In addition, Bacillus subtilis mutant CJKB0011 according to the present invention has the characteristics of producing riboflavin at a concentration of 16.3% higher than the parent strain Bacillus subtilis CJKB0001 strain during the culture of the flask for riboflavin production, 51 in culture fermenter It is characterized by producing riboflavin at a concentration of about 19.4% higher than the strain.

Therefore, Bacillus subtilis mutant CJKB0011 according to the present invention is a strain of fermentation by-product acetoine production is significantly lowered to 26.2%, and riboflavin production ability is significantly improved.

The present invention also provides a method for producing riboflavin from the culture by culturing Bacillus subtilis mutant CJKB0011 according to the present invention under suitable conditions.

That is, the Bacillus subtilis mutant CJKB0011 (KCCM-10525) is inoculated in a conventional medium containing a suitable carbon source, nitrogen source, amino acids and vitamins, and cultured by controlling 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 5 to 6 days.

In one embodiment according to the present invention inoculated Bacillus subtilis mutant CJKB0011 of the present invention in a seed medium and incubated for 20 hours at 37 ℃, 800rpm while supplying air at 1vvm, inoculated the seed culture solution in fermentation medium by air Is supplied 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 is 0.5-1%. Riboflavin was obtained at a concentration of about 19.4% 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 CJKB0011 according to the present invention>

Bacillus subtilis mutant strain CJKB0011 of the present invention is treated with a mutagen Bacillus subtilis CJKB0001 induction of mutations and then cultured in a minimal medium to obtain a colony capable of growth and then inoculated in MR-VP broth Subsequently, a portion of the culture solution was taken to perform Voges-Proskauer test to select low-producing acetoin strains, and to select mutants having the best riboflavin-producing ability among them.

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 (N-methyl-N'-nitro-N-nitroguanidine), was added to a final concentration of 10-50 µg / ml. This was treated for 30 to 60 minutes at room temperature or 30 ° C. to induce mutations. This was washed three times with 0.85% saline, diluted appropriately, and then plated in a minimal medium containing 1.8% agar, followed by incubation at 37 ° C. for 24 hours to obtain colonies. The obtained colonies were inoculated in 5 ml of MR-VP broth and incubated at 35 ° C. for 24 hours. Thereafter, 1 ml of the culture solution was taken and the Voges-Proskauer test was performed to observe the change in chromaticity by adding 0.6 ml of 5% α-naphtol and 0.2 ml of 40% KOH. As a result, the strains with the smallest color change were selected, inoculated into fermentation broth and incubated at 37 ° C. for 4 to 5 days to select the best strains of riboflavin accumulated in the fermentation broth. The amount of riboflavin produced was analyzed using HPLC. The analysis method of riboflavin using HPLC is as follows.

Device: Waters 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.

    Composition of medium for the selection of acetoin low producing strain badge Furtherance Nutrition medium Trypsose 10g / l, Beef Extract 3g / l, Sodium Chloride 5g / l, Agar 15g / l, pH 7.2 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 20 mg / l, pH 7.2 MR-VP broth Polypeptone 7 g / l, glucose 5 g / l, dipotassium phosphate 5 g / l, pH 7.0 Fermentation medium Glucose 100g / l, Yeast extract 20g / l, Corn steep 5g / l, Magnesium sulfate 0.5g / l, Potassium phosphate 1.5g / l, Dipotassium phosphate 3.5g / l, Urea 6g / l, pH 7.2

 As a result, acetoin-producing mutants had excellent riboflavin production capacity. Among them, one strain of the best strain of riboflavin production was selected and named as Bacillus subtilis strain CJKB0011. The selected Bacillus subtilis mutant strain CJKB0011 was deposited with the Korean Culture Center of Mocroorganisms on November 17, 2003 and was assigned accession number KCCM-10525.

<Example 2: Comparison of acetoin generating ability of Bacillus subtilis mutant CJKB0011 and Bacillus subtilis CJKB0001 used as a parent strain according to the present invention>

Bacillus subtilis mutant strain CJKB0011 (KCCM-10525) selected in Example 1 and Bacillus subtilis CJKB0001 used as parent strains were cultured in a 51 fermenter to compare acetoin production ability. 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 1 l each in a 2.5L experimental fermenter and then prepared by autoclaving at 121 ° C for 10 minutes. After cooling it, the Bacillus subtilis mutant CJKB0011 and the parent strain according to the present invention were suspended in saline and inoculated with 10 ml, and then inoculated with 1vvm of sterilized air and incubated for 20 hours with stirring at 8000 rpm at 37 ° C. The pH was not adjusted during incubation. When the seed culture was completed, the fermentation medium was inoculated to perform fed-batch fermentation. Fermentation medium was prepared by dispensing 1.41 each in 51 volumes of experimental fermenter and autoclaving at 121 ° C. for 20 minutes. After cooling it, the seed culture solution was inoculated with 200 ml and incubated at 8000 rpm and 40 ° C. while supplying 1vvm of air. At this time, an additional medium was supplied so that the residual sugar concentration was 0.5-1% during the cultivation so that the total sugar concentration added to the fermentation medium was 20%. The pH of the culture was incubated for 60 ~ 70 hours, adjusting to 7.0 using ammonia water. The composition of the medium used in the present invention is as shown in Table 4. The amount of acetoin produced was analyzed by gas chromatography. The analysis method of acetoin using gas chromatography is as follows.

Equipment: Gas Chromatography HP-6890 Series II

      (gas chromatography HP-6890 series II)

Column: HP-FFAP, FUSED SILICA Capillary column

Length: 30m, Inner diameter: 1.0um

Carrier Gas: Helium Flow Rate: 9.5ml / min

As a result, as shown in Table 2, Bacillus subtilis CJKB0001 strain used as a parent strain produced about 4.2 g / l of acetoin as a fermentation by-product after 65 hours of incubation in a 51 fermenter. In contrast, Bacillus subtilis mutant CJKB0011 according to the present invention produces about 1.1 g / l of acetoin under the same conditions, and has a low acetoin production capacity of about 26.2% compared to the acetoin production capacity of the parent strain. Confirmed.

Comparison of Acetoin Formation Capacity of Bacillus Subtilis Mutant CJKB0011 and Bacillus Subtilis CJKB0001 Strain / concentration (g / l) 0 19 hours 40 hours 52 hours 60 hours 65 hours Bacillus subtilis CJKB0001 0 0.4 One 1.8 3.0 4.2                                              Bacillus subtilis CJKB0011 (KCCM-10525) 0 0.13 0.24 0.46 0.76 1.1                                             

<Example 3: Flask fermentation titer of Bacillus subtilis mutant CJKB0011 according to the present invention>

The flask fermentation titer of Bacillus subtilis CJKB0011 (KCCM-10525) 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. Here, inoculated with the mutant strain and the parent strain according to the present invention and incubated for 20 hours at 200rpm, 37 ℃. 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 hours. The flask fermentation medium was prepared by autoclaving the medium and starlet medium in the same manner as the seed medium, and then dispensing 15 ml and 5 ml, respectively, in a 250 ml shake flask that had been autoclaved in advance. 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: 1.5 g / l potassium phosphate, 3.5 g / l potassium phosphate, urea 6 g / l, pH 7.2 ~ 7.4

As a result, the production of riboflavin in the medium of the parent strain Bacillus subtilis CJKB0001 was 8.0g / l, the production of riboflavin in the medium of the Bacillus subtilis mutant CJKB0011 according to the present invention was 9.3% improved by a whopping 16.3% g / l.

<Example 4: Fermentation titer in 51 fermenter of Bacillus subtilis mutant CJKB0011 according to the present invention>

 The fermentation titer of 51 Bacillus subtilis CJKB0011 (KCCM-10525) 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 it, the Bacillus subtilis mutant CJKB0011 and the parent strain of the present invention were suspended in saline, respectively, and inoculated by 10 ml, and then inoculated with sterilized air at 1vvm and incubated for 20 hours while stirring at 8000 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 51 volumes of 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 8000 rpm, 40 ℃ 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 medium was 20%. The pH of the culture was incubated for 60 ~ 70 hours, adjusting to 7.0 using ammonia water.

At this time, glucose was replaced with molasses as a carbon source in seed and fermentation broth, and yeast extract and tryptone were replaced with corn steep liquor as a nitrogen source. Additional medium for fed-batch fermentation was glucose as a carbon source and dry yeast and corn steep liquor as a nitrogen source. 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, potassium phosphate 1.5 g / l, phosphate 2 Potassium 3.5g / l, pH 7.4 Fermentation medium Main medium: 20g / l dry yeast, 5g / l corn steep liquor, 2g / l ammonium sulfate, 0.5g / l magnesium sulfate heptahydrate, 17.5g / l potassium phosphate, 7.5g / l potassium diphosphate, pH 7.2 ~ 7.4 Additional medium: glucose 270g / l, dry yeast 26.7g / l, corn steep 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 CJKB0011 according to the present invention, it was 32.0 g / l which was improved by about 19.4% compared to the parent strain.

As described above, the Bacillus subtilis mutant CJKB0011 according to the present invention generates acetoin, which is a major fermentation byproduct, at a very low level compared to the parent strain when glucose is used as a carbon source. There is an effect that can be produced in yield. 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 CJKB0011 KCCM-10525, characterized in that the ability to produce acetoin, a fermentation by-product produced during the fermentation process to produce riboflavin, is reduced than that of the parent strain Bacillus subtilis CJKB0001 KCCM-10445. . delete A method of producing a riboflavin from the culture by culturing the microorganism according to claim 1.