KR20050055176A - Microorganism producing riboflavin and method for producing riboflavin using thereof - Google Patents

Abstract

The present invention relates to Bacillus subtilis strain CJKB0005 using a genetic recombination technique and a method for producing riboflavin using the microorganism. The present invention increases the amount of riboflavin by increasing the expression level of phosphoribosyl pyrophosphate synthetase, an enzyme that synthesizes 5-phosphoribosyl 1-pyrophosphate, an intermediate step of riboflavin production. It is characterized by increasing the. Increased expression of phospholibosyl pyrophosphate synthetase enhances the metabolic flow of the pentose phosphate pathway, which in turn facilitates the supply of precursors necessary for riboflavin production, thus ultimately producing high concentrations of riboflavin Done. Therefore, the present invention has the effect of culturing the microorganism and obtaining a large amount of riboflavin from the culture.

Description

Microorganisms producing riboflavin and 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 Bacillus subtilis recombinant strain CJKB0005 and riboflavin are produced using the microorganism, characterized by enhancing the pentose phosphate pathway through an increase in the expression level of phosphoribosyl pyrophosphate synthetase, thereby improving riboflavin production. It is about how to.

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.

Phosphoribosyl pyrophosphate synthetase, on the other hand, is an enzyme that catalyzes a reaction that attacks the beta-phosphoryl group of the nucleoside triphosphate chain. The resulting phosphoribosyl pyrophosphate acts as an important precursor in the reaction pathways such as nucleotide biosynthesis, nucleobase regeneration, pyrimidine coenzyme production, tryptophan and histidine biosynthesis.

Phosphoribosyl pyrophosphate synthetase, which functions as described above, is a vector for Bacillus in order to produce a large amount of nucleic acid-related substances such as inosine or guanosine, as disclosed in Japanese Patent Application Laid-Open No. 5-192164. It has been reported to use recombinantly.

 The inventors of the present invention, while studying the microorganisms with improved production of riboflavin, by overexpressing the phosphoribosyl pyrophosphate synthetase in Bacillus to enhance the nucleic acid production pathway and eventually create a strain that produces a high concentration of riboflavin Completed.

It is therefore an object of the present invention to provide a Bacillus subtilis recombinant strain CJKB0005 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 recombinant strain CJKB0005 (KCCM-10527) to produce riboflavin.

The present invention also provides a method for producing riboflavin using the Bacillus subtilis recombinant strain CJKB0005.

Hereinafter, the present invention will be described in detail.

Bacillus subtilis recombinant strain CJKB0005 (KCCM-10527) according to the present invention is a recombinant strain that produces riboflavin, because it overexpresses phosphoribosyl pyrophosphate synthetase, thereby producing a large amount of nucleic acid-related substances, and eventually riboflavin at a high concentration. There is a characteristic to produce.

Based on the riboflavin synthesis pathway, macroarray results of the Bacillus subtilis mutant CJKB0001 show that compared to the Bacillus subtilis wild strain, it is an enzyme that synthesizes phosphoribosyl pyrophosphate, an intermediate of the riboflavin synthesis step. The expression of poribosyl pyrophosphate synthetase was found to be reduced by half.

Therefore, the present inventors cloned the phospholibosyl pyrophosphate synthetase of wild strains of Bacillus subtilis into a vector that can be used in Bacillus, introduced them into the parent strain CJKB0001, and selected the best strains of riboflavin production among them. .

The vector used was a vector made by inserting a puromysin resistance gene into the kanamycin resistance gene gene position among the antibiotics of pDG148, which was named pDPu6.

Bacillus subtilis used as the parent strain was used strain CJKB0001 (Korean Patent No. 10-2002-076868) having resistance to the proline analog.

The inserted gene, phosphoribosyl pyrophosphate synthetase, was prepared in two forms. First, the promoter and phosphorinebolic pyrophosphate synthetase are expressed by using a promoter and sine-dalgarno sequence of phosphoribosyl pyrophosphate synthetase itself. It was designed to be expressed by combining the sine-dalgano sequence of Tase itself.

The present inventors cloned the phosphoribosyl pyrophosphate synthetase gene obtained by polymerase chain reaction (PCR) from the chromosome of the Bacillus subtilis wild strain to the Sal I and BamH I sites of pDPu6, and then, pDP- P1 and pDP-P2 were prepared, and the prepared plasmid vector was transformed into Bacillus subtilis CJKB0001 again. The selection of recombinant strains was confirmed by separating the plasmids of the strains using polymerase chain reaction and restriction enzyme digestion. The recombinant strains thus obtained were compared with the parent strain in the Erlenmeyer flask containing the titer medium (Table 1). As a result, the parent strain produced 8.0 g / l at 96 hours, whereas the phospholibosyl pyrophosphate synthetase Strain CJKB0005, the best performing recombinant strain with enhanced expression, achieved a yield improvement of about 12.5% by producing 9.0 g / l riboflavin. In addition, 51 strains of fermenter produced 29.5 g / l riboflavin with a 10.1% improvement over the parent strain CJKB0001 (26.8 g / l).

Therefore, Bacillus subtilis recombinant strain CJKB0005 according to the present invention is a strain in which the expression level of phosphoribosyl pyrophosphate synthetase is increased in comparison with the parent strain, and the riboflavin synthesizing intermediate step material is smoothly supplied and the riboflavin production ability is remarkably improved. to be.

The present invention also provides a method for producing riboflavin from the culture by culturing the Bacillus subtilis recombinant strain CJKB0005 according to the present invention under suitable conditions.

That is, the Bacillus subtilis recombinant strain CJKB0005 (KCCM-10527) 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 2 to 6 days.

In one embodiment according to the present invention inoculated Bacillus subtilis recombinant strain CJKB0005 of the present invention to the seed medium and incubated for 20 hours at 37 ℃, 800rpm while supplying air at 1vvm, and then inoculated the seed culture solution to the fermentation medium While supplying air at 1 vvm and shaking incubation at 40 ° C., 800 rpm, and pH 7.0 for 60 to 70 hours, an additional medium containing glucose was added so that the residual sugar concentration in the culture was 0.5 to 1%. Riboflavin was obtained at a concentration of about 10.1% improved compared to the parent strain by culturing by addition to%.

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: Preparation of the primer for the acquisition of phosphoribosyl pyrophosphate synthetase according to the present invention>

In order to obtain the gene required for the recombinant strain CJKB0005 of the present invention, a primer was prepared for using the phosphoribosyl pyrophosphate synthetase gene of the wild strain as a template. Restriction enzyme sequences were prepared before and after the gene to be PCR for insertion into a vector that can be used in Bacillus. The front part of the gene was Sal I, the back part was BamH I, and the DNA fragments were linked to each other, including the sequence of Kpn I. Restriction enzyme sequences are underlined. Figure 1 shows a primer preparation for obtaining phosphoribosyl pyrophosphate synthetase.

prs-1 (Sal I) gat aga cgc gtc gac gaa ttc gaa gaa gct gga

prs-2 (Kpn Ⅰ) cgt cgg ggt acc aaa ccg ctt atc cat tta

prs-3 (Kpn Ⅰ) cat cgg ggt acc att cat aaa aaa taa tcc

prs-4 (BamH Ⅰ) cgt cgc gga tcc aat ggt tta gct gaa cag ata

prs-3 '(Sal Ⅰ) gat aga cgc gtc gac taa tcc taa att cgg agg ttt

<Example 2: Phosphoribosyl pyrophosphate synthetase coding and gene acquisition according to the present invention>

The polymerase chain reaction was performed using pfu polymerase (stratagene) as a template using chromosomal DNA of the Bacillus subtilis wild strain using the primer prepared in Example 1. The conditions were carried out for 30 seconds at 94 ° C denaturation step, 1 minute at 55 ° C annealing step, and 1 minute 30 seconds at 72 ° C elongation step, 28 Was performed twice. The primer sets used were prs-1 (Sal I) & prs-2 (Kpn I) <Reaction 1>, prs-3 (Kpn I) & prs-4 (BamH I) <Reaction 2>, prs-3 '( Sal I) & prs-4 (BamH I) <Reaction 3>. The reaction products 1, 2, and 3 obtained after the polymerase chain reaction were found to be about 0.2 kb, 1.0 kb, and 1.0 kb. The results of 1 and 2 were cut with the restriction enzyme Kpn I and conjugated with T4 DNA ligase. The conjugated 1.2 kb of DNA was used as a template, and primers prs-1 (Sal I) & prs-4 ( Using BamH I) <Reaction 4>, the polymerase chain reaction was carried out in the same manner as above to obtain 1.2 kb of the product.

Example 3 Assembly of Phosphoribosyl Pyrophosphate Synthetase Gene and Vector According to the Present Invention

After the results of reactions 3 and 4 obtained in Example 2 were cut with restriction enzymes Sal I and BamH I. After electrophoresis on 0.7% agarose gel, a band of the desired size was eluted. Each resultant was cut into Sal I and BamH I and then conjugated with pDPu6 vector digested with Sal I and BamH I at 16 ° C. for 24 hours. The recombinant plasmids were transformed into Escherichia coli DH5α, incubated for 24 hours at 37 ° C. in a solid medium containing Ampicillin 100mg / l. Plasmid DNA was isolated using a QIAGEN miniprep kit. The plasmid into which the resultant obtained in reaction 4 was introduced was named pDP-P1 and the resultant obtained in reaction 3 was named pDP-P2.

Example 4: Identification of assembled pDP-P1 and pDP-P2 plasmids according to the present invention and preparation of CJKB0005

As shown in Fig. 2, in order to confirm the plasmid DNAs of pDP-P1 and pDP-P2 isolated in Example 2, the plasmid was used as a template, and primer sets prs-1 (Sal I) & prs-4 (BamH I ), prs-3 '(Sal I) & prs-4 (BamH I) confirmed that the results were 1.2kb, 1kb sized. And it was confirmed that the gene introduced into the assembled plasmid was also cut using restriction enzymes Sal I and BamH I. Each identified pDP-P1, pDP-P2 plasmid was transformed into the parent strain CJKB0001. The strain into which the plasmid pDP-P1 was introduced was named CJKB0005, and the strain into which pDP-P2 was introduced was named CJKB0006. Among the microorganisms, Bacillus subtilis recombinant strain CJKB0005 was deposited with the Korean Culture Center of Microorganisms on November 17, 2003 and received accession number KCCM-10527.

<Example 5: Flask fermentation titers of Bacillus subtilis recombinant strains CJKB0005 and CJKB0006 according to the present invention>

The flask fermentation titer of Bacillus subtilis CJKB0005 according to the present invention was investigated in comparison with the fermentation titer of Bacillus subtilis CJKB0001, the parent strain. Recombinant strains and parent strains according to the present invention were seeded and seeded in the seed medium, respectively, followed by fermentation by seeding the culture medium with 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. Herein, the recombinant strains and the parent 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 concentration was analyzed by HPLC, in this case the analysis method 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

The composition of the seed medium and fermentation medium used in the experiment is shown in Table 1.

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 1, 3.5g / l potassium phosphate dibasic, 6g / l urea, erythromycin 10mg / l, chloramphenicol 10mg / l, puromycin 20mg / 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, while the riboflavin accumulation in the medium of the Bacillus subtilis recombinant strain CJKB0005 according to the present invention was improved by 12.5% compared to the parent strain. 9.0 g / l. In the case of CJKB0006, it was 8.3g / l, an improvement of 3.6%.

Example 6: Fermentation titers of Bacillus subtilis recombinant strains CJKB0005 and CJKB0006 according to the present invention

The fermentation titer in the 51 fermenter of Bacillus subtilis CJKB0005 according to the present invention was investigated in comparison with the fermentation titer of Bacillus subtilis CJKB0001, the parent strain. The recombinant strain and the parent strain according to the present invention were inoculated in a seed medium, followed by seed culture, and then seed culture was inoculated in a fermentation medium to perform fed-batch fermentation. First, the seed medium was dispensed by 11 into 2.5l fermentation tank for experiment, and then prepared by autoclaving at 121 ° C for 10 minutes. After cooling, the Bacillus subtilis recombinant strain and the parent strain according to 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 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 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 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 medium was 20%. 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 method by HPLC is as described above.

 The composition of the medium used in the present invention is shown in Table 2.

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 3.5 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, whereas the recombinant strain CJKB0005 according to the present invention was 29.5 g / l, which was about 10.1% improved compared to the parent strain. In the case of CJKB0006, it was 27.2 g / l, which was about 1.1% more.

As described above, in the Bacillus subtilis recombinant strain CJKB0005 according to the present invention, the expression of phosphoribosyl pyrophosphate synthetase is increased compared to the parent strain CJKB0001, thereby enhancing the intracellular pentose phosphate pathway. As a result, nucleic acid-related substances, which are riboflavin intermediates, are rapidly generated, and thus, riboflavin can be produced at a high concentration. Therefore, the present invention has the effect of culturing the microorganism and obtaining a large amount of riboflavin from the culture.

1 is a diagram for preparing a primer for obtaining phosphoribosyl pyrophosphate synthetase.

2 is a view showing the manufacturing process of the pDP-P1, pDP-P2 plasmid according to the present invention.

Claims (4)

Bacillus subtilis produced with riboflavin and transformed with a vector comprising a phosphoribosyl pyrophosphate synthetase gene. The vector pDPu6 according to claim 1, wherein the vector comprises a phosphoribosyl pyrophosphate synthetase gene and a vector made by inserting the puromycin resistance gene into the kanamycin resistance gene position of pDG148. Bacillus subtilis, characterized in that pDP-P1 or pDP-P2 prepared using. The method of claim 2, Bacillus subtilis CJKB0005 KCCM-10527 transformed with recombinant vector pDP-P1. A method of culturing a microorganism according to any one of claims 1 to 3 to produce riboflavin from the culture.