KR101431453B1 - Biological method for efficient producing 5-aminolevulinic acid, porphyrin, porphyrin derivative or heme - Google Patents

Abstract

The present invention relates to a biological method for the efficient production of 5-aminolevulinic acid, porphyrin, porphyrin analogue or heme, and more particularly to a method for the production of 5-aminolevulinic acid, porphyrin, Aminolevulinic acid, porphyrin, porphyrin analogs or heme, characterized in that a medium having a composition with enhanced pH stabilizing ability is used which is characterized by the addition of a 5-aminolevulinic acid, porphyrin analog or heme.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a biological method for efficiently producing 5-aminolevulinic acid, porphyrin, porphyrin derivative or heme,

The present invention relates to a biological method for the efficient production of 5-aminolevulinic acid, porphyrin, porphyrin analogue or heme, and more particularly to a method for the production of 5-aminolevulinic acid, porphyrin, Aminolevulinic acid, porphyrin, porphyrin analogs or heme, characterized in that a medium having a composition with enhanced pH stabilizing ability is used which is characterized by the addition of a 5-aminolevulinic acid, porphyrin analog or heme.

Heme (heme) iron ions (Fe ^{2 +),} a porphyrin (porphyrin) complex (complex), and sseoksi carbonyl containing-path by the polymerization of Koei (succinyl-CoA), and glycine (glycine) (C4 path) Or glutamate as a starting material, through the pathway (C5 pathway) using ATP, NADPH coenzyme. The preferences for these two paths vary from species to species. Two paths both 5-aminolevulinic acid synthase, called acid 5-amino-LES by the action of (5-aminolevulinic acid synthase; hem A; ALAS); and starting from the synthesis of (5-aminolevulinic acid ALA), the Fe2 + ferro Mosquera other kinase; (; Proto IX protoporphyrin IX) synthesis of heme being inserted (integration) in this is completed (ferrochelatase hem H) protoporphyrin IX by (Appl Microbiol Biotechnol 63: 115-127, 2003...).

In the process of hem biosynthesis, various porphyrin derivatives are produced. The porphyrin analogs include porphobilinogen (PBG), 1-hydroxymethylbilane (HMB), uroporphyrin I (Uro I), uroporphyrin III (Uro III) Coproporphyrin I (Copro I), coproporphyrin III (Copro III), and protoporphyrin IX. The hem biosynthetic pathway is schematically shown in Fig.

Heme plays an important role in oxygen transfer, removal of reactive oxygen, and electron transfer related to energy production (Cell 122: 487-489, 2005). Among the porphyrin analogues and complexes, heme is of particularly high commercial significance. Hem can be used as an organic iron source. The bio-absorption rate of the organic form of iron is twice as high as that of the inorganic form of iron and is recognized as a better source of iron (Crit. Rev. Food Sci. Nutr. 31: 333-367, 1992; Acta. Med. Scand 629: 1-46, 1980). For this reason, hem is very likely to be used as an iron supply agent or anemia treatment agent. It can also be used as an iron feeder or feed additive in the livestock sector.

5-Aminolevulinic acid is also important in industrial applications. 5-Aminolevulinic acid has been used as an environmentally friendly biocide (photoactive herbicide) and plant growth promoter. When 5-aminolevulinic acid is treated at a low concentration, radish, kidney bean, barley, potato, garlic, rice, corn (Plant Growth Regulation 22: 109-114, 1997), compared with the untreated group. It can also be used in the pharmaceutical sector. 5-Aminolevulinic acid is currently being used as a treatment for skin diseases such as acne, atopy and skin cancer, and it is also used in cosmetics. In the livestock sector, it can also be used as a growth promoter for antibiotic substitution for the purpose of enhancing the autoimmunity of livestock and improving feed efficiency (Korean Patent Registration No. 10-0459918).

On the other hand, various other porphyrin analogues or complexes whose industrial applications are not yet clear are expected to be followed by industrial applications as their physiological activities are gradually identified.

However, the development of a biological method capable of efficiently producing 5-aminolevulinic acid, porphyrin, porphyrin analog or heme has not been developed yet. Although the method of biological production of heme has been disclosed by the present inventors (Korean Patent Laid-Open Publication No. 10-2011-0070977), since this method uses a complex medium in the production of heme, economical efficiency And there is also a difficulty in controlling the hem production due to the characteristics of the complex media components.

It is therefore necessary to develop a synthetic medium suitable for industrial production of heme (also referred to as chemically defined medium, chemically defined medium, chemically defined medium, or chemically defined medium, often referred to in English as chemically defined medium) And these media can be utilized in the production of 5-aminolevulinic acid, porphyrin or porphyrin analogs, which are expected to have various industrial applications besides heme.

Numerous papers and patent documents are referred to throughout this specification and the citations are indicated in parentheses. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

SUMMARY OF THE INVENTION Accordingly, the present invention has been made to solve the above-mentioned problems occurring in the prior art and the technical problems required from the past.

It is an object of the present invention to provide a biological method for the efficient production of 5-aminolevulinic acid, porphyrin, porphyrin analogs or heme.

It is yet another object of the present invention to provide a synthetic medium composition suitable for biological methods for the efficient production of 5-aminolevulinic acid, porphyrin, porphyrin analogs or heme.

As a result of various efforts, the inventors of the present invention have made efforts to solve the above-mentioned problems, and as a result of various efforts, the present inventors have found that a pH Confirming that the synthetic medium composition with enhanced stabilization ability is more suitable for the biological production of 5-aminolevulinic acid, porphyrin, porphyrin analog or heme.

The addition of bicarbonate helps to synthesize several precursors participating in the C4 pathway as a component added to enhance the heme biosynthetic C4 pathway. In particular, bicarbonate acts as a substrate in the production of CO ₃ ^- required for the activity of the NADP-dependent malic enzyme ( mae B), an important enzyme in heme biosynthesis (J. Appl. Microbiol, 103: 2340-2345, 2007). Although embodiments of the present invention disclose the use of sodium bicarbonate, various salt-type bicarbonates are obviously applicable. The amount of the bicarbonate to be used may vary depending on the type of the salt used, and it is preferable to use from 0.01 g / L to 50 g / L, more preferably from 2 g / L to 20 g / L .

Alternatively, a method of supplying a mixed gas containing carbon dioxide gas or carbon dioxide gas may be substituted for the addition of bicarbonate. At this time, the carbon dioxide content of the mixed gas is preferably 0.5-30%, more preferably 1-10%.

The addition of the sodium decanate is a component added to induce the activation of the heme biosynthetic C4 pathway, and is a component utilized as a starting material for the rotynil-koa of the C4 biosynthesis C4 pathway. Although embodiments of the present invention disclose the use of sodium regrindate, it is apparent that various salt salt rotundans are applicable. It is preferable to use the above-mentioned decanate from 0.01 g / L to 50 g / L, more preferably from 2 g / L to 20 g / L.

The addition of glycine is a component added to induce the activation of the heme biosynthetic C4 pathway, which participates in the polymerization of the rotycin-nose of the heme biosynthetic C4 pathway. The glycine is preferably used in an amount of 0.01 g / L to 20 g / L, more preferably 1 g / L to 5 g / L.

The growth factor refers to a component that provides an effect of increasing the concentration of intracellular metabolites or coenzyme species necessary for heme biosynthesis or may act as a component promoting heme biosynthesis in itself, and vitamins are suitable. Various growth factors may be used, and it is preferable that the growth factor is selected from thiamin, biotin, nicotinic acid, pantothenic acid, cobalamin (also called vitamin B12) It is most preferable to use all of them. These growth factors are preferably used at an individual concentration of from 0.001 mg / L to 10 mg / L, and more preferably from 0.005 mg / L to 5 mg / L.

The enhancement of the pH buffering capacity is necessary to alleviate the effect of the change in pH of the medium due to the consumption of added bicarbonate, which can be achieved by adding a high concentration of the pH buffer component. The present inventors have found that the production of heme is seriously inhibited when the pH of the culture medium is lowered due to the consumption of bicarbonate. The concentration of the buffer solution may be 10-500 mM, but it is preferably 50-500 mM and more preferably 100-500 mM for a definite effect.

The buffer is not limited as long as it is a buffer capable of providing a pH buffering effect, but it is preferable to use a phosphate buffer.

Using the medium composition of the present invention, the control over the biological production of 5-aminolevulinic acid, porphyrin, porphyrin analog or heme can be more thoroughly controlled, and the economical efficiency of the production process can be further improved.

Figure 1 is a schematically illustrated hem biosynthetic pathway.
Figure 2 is a graph showing the synthesis of porphyrin analogs.
Figure 3 shows the results of LC / MS analysis confirming the production of heme.
Figure 4 shows the results of LC / MS analysis confirming the production of 5-aminolevulinic acid.
FIG. 5 shows the results of LC / MS analysis confirming the production of porphyrin III.
Fig. 6 shows the results of LC / MS analysis confirming the production of coproporin III.
Fig. 7 shows the result of LC / MS analysis confirming the production of protoporphyrin IX.
FIG. 8 shows the result of the analysis according to the incubation time. (A) 5-aminolevulinic acid; (B) a phospholipids; (C) Uroporphyrin I; (D) Coproporphyrin I; (E) coproporphyrin III; (F) heme; And (G) cell concentration.

As described above, the present invention provides a synthetic medium composition suitable for a biological method capable of efficiently producing 5-aminolevulinic acid, porphyrin, porphyrin analog or heme.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are merely examples of the present invention, and the scope of the present invention is not limited to these Examples.

Example  1: Using Production strain

As a production strain of 5-aminolevulinic acid, porphyrin, porphyrin analog or heme of the present invention, a strain prepared by the present inventors and disclosed in Korean Patent Publication No. 10-2011-0070977 was used. This strain is a strain produced by transforming E. coli W3110 with ^{pTrc (PlachemA + -maeB-dctA)} , 5- aminolevulinic acid synthase (5-aminolevulinic acid synthase; hem A), NADP- dependent malic enzyme (NADP -dependent malic enzyme ( mae B), and a dicarboxylate transporter ( dct A), which can be used for production through biological methods of heme.

Example  2: Usage badge

The medium used in the present invention has a medium composition modified from the medium composition known as M9 medium. The greatest feature of the medium composition of the present invention is that it contains bicarbonate, roticinate and glycine. In addition, the growth factors include a group of vitamins selected from thiamin, biotin, nicotinic acid, pantothenic acid, cobalamin, and the like, and further include a pH buffer component having a high concentration. The composition of the medium used in this example is shown in Table 1 below.

ingredient Content or amount added NaHCO ₃ 10 g / L Glucose 10 g / L NH ₄ Cl 0.2 g / L NaCl 0.1 g / L Glycine 2 g / L Disodium succinate hexahydrate 10 g / L CaCl ₂ 0.015 g / L MgSO ₄ .7H ₂ O 0.246 g / L Ampicillin 20 μg / ml Trace element solutions (see Table 2 below) 200 μl / L Vitamin solutions (see Table 3 below) 1 ml / L The pH stabilizing solution (see Table 4 below) 500 ml / L IPTG 0.1 mM

ingredient content Citric acid 50 g / L ZnSO ₄ .7H ₂ O 50 g / L FeSO ₄ .7H ₂ O 47.5 g / L _{Fe (NH 4) 2 (SO} 4) 2 6H ₂ O and 10 g / L CuSO ₄ .5H ₂ O 2.5 g / L MnSO ₄ .H ₂ O 0.5 g / L H ₃ BO ₃ 0.5 g / L Na ₂ MoO ₄ .2H ₂ O 0.5 g / L CoCl ₂ .2H ₂ O 0.5 g / L NiSO ₄ .6H ₂ O 0.5 g / L

ingredient content Thiamin ㆍ HCl 1 mg / L Nicotinic acid 5 mg / L Pantothenic acid 1 mg / L Biotin 0.01 mg / L Cobalamin 1.4 mg / L

ingredient Addition amount (1 L) 0.2 M Na ₂ HPO ₄ solution 333.3 ml 0.2 M KH ₂ PO ₄ solution 666.7 ml

In addition, a culture medium in which neither the decanate nor glycine was added was also prepared. This was used in a negative control experiment.

On the other hand, a method of supplying carbon dioxide gas instead of adding bicarbonate was considered to provide the same technological effect. To carry out the investigation, a medium containing the same except for the bicarbonate was prepared.

Example  3: Culture method

Production of 5-aminolevulinic acid, porphyrin, porphyrin analogs or heme was carried out in the following manner. A single colony of the production strain was inoculated into a 15 ml tube containing 3 ml of LB (Luria-Bertani) medium and incubated for 16 hours at 37 DEG C with stirring at 250 rpm. The thus cultured medium was inoculated to a medium containing up to the bicarbonate shown in Example 2 at a volume ratio of 5%, followed by culturing. This culture was also carried out for 48 hours in the manner of stirring at 37 ° C and 250 rpm.

As a further experiment, experiments were conducted in which the main culture was carried out in the same manner using the medium in which bicarbonate was excluded from the Example 2. At this time, a mixed gas containing carbon dioxide (21% oxygen, 3% carbon dioxide, 76% nitrogen) was supplied at a rate of 2 L / min to compensate for the role of the excreted bicarbonate.

Example  4: Analysis method

The cell density was determined by measuring the absorbance at 600 nm using a spectrophotometer according to a conventional method.

Analysis of porphyrin analogs containing heme was carried out as follows. For analysis of 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), 1 ml of the culture broth was centrifuged at 13,000 rpm for 5 minutes at 4 ° C to recover the supernatant Respectively. ALA and PBG were separated from the recovered supernatant by ion-exchange chromatography using an ALA / PBG column test kit (Bio-Rad Laboratories, Hercules, Calif., USA). The amount of separated ALA and PBG was determined by reacting with Ehrlich's reagent according to the reported method (Int. J. Biochem. Cell Biol. 30: 535-543, 1998), and then measuring the absorbance at 550 nm .

Analysis of heme was carried out as follows. 1 ml of the culture was centrifuged (at 4 ° C for 13 minutes at 13,000 rpm for 5 minutes), and then the supernatant was removed to recover only the cells (cells). The recovered cells were suspended in 200 μl of 1 N NaOH solution. Cell suspension was sonicated at 30 W for 20 minutes at 0 ° C using an ultrasonic shaker (UP200S sonicator, Hielscher Ultrasonic GmbH, Teltow, Germany) for 20 minutes to disrupt the cells. The cell lysate was centrifuged at 13,000 x g for 10 minutes at 4 ° C to obtain supernatant. 200 μl of the recovered supernatant was mixed with 400 μl of a 4: 1 mixed solution of acetonitrile and dimethylsulfoxide (DMSO). The mixture was vigorously mixed and centrifuged at 13,000 × g for 10 minutes at 4 ° C. to separate into two layers. Each layer was recovered and subjected to high performance liquid chromatography (HPLC) analysis. HPLC analysis was carried out by reverse-phase HPLC (RP-HPLC) using an octadecylsilyl CIS column (Xbridge C18, Waters). The solvent used as mobile phase in HPLC analysis was a solution prepared by mixing 86% methanol in 1 M ammonium acetate buffer adjusted to acetic acid to pH 5.16 by volume ratio. The mobile phase solvent was flowed at an isocratic elution rate of 1 ml / min. Detection was carried out by measuring the absorbance at 400 nm using a UV detector (2489 UV / visible detector; Waters). Hemin (hemin; Sigma-Aldrich, St. Louis, Mo., USA) was used as a reference material in this analysis and a heme was quantified using a standard curve.

The analysis of porphyrin I, coproporphyrin I and coproporphyrin III was carried out as follows. 1 ml of the culture was centrifuged at 13,000 x g for 10 minutes at 4 ° C to recover the supernatant. 200 μl was taken from the recovered supernatant and mixed with the same volume of cold acid-acetone (refrigerated with acetone mixture containing 0.2% 10 N HCl in a volume ratio). The mixed solution was subjected to heat treatment in a water bath for 30 minutes at 65 ° C to denature the proteins contained in the recovered supernatant. The denatured proteins were removed by centrifugation at 13,000 x g for 10 minutes at 4 ° C. The supernatant obtained after centrifugation was subjected to vacuum drying. The powder remaining after vacuum drying was dissolved by adding 200 μl of 1 N NaOH solution. Reversed phase HPLC analysis was performed using the thus obtained solution as a sample. As the HPLC column, an octadecylsilyl CIS column was used, and solvent A (water containing 0.05% of trifluoroacetic acid) and solvent B (acetonitrile containing 0.05% of trifluoroacetic acid) ) In a concentration gradient manner. The application of the concentration gradient was carried out by flowing 100% solvent A for the first 5 minutes and then increasing the solvent B at a volume ratio of 0-50% for 25 minutes thereafter. In the last 10 minutes, the solvent B And the flow rate was increased. The mobile phase solution was flowed at a rate of 1 ml / min. Detection was carried out by measuring the absorbance at 400 nm using a UV detector (2489 UV / visible detector; Waters).

For the identification of porphyrin analogues, LC / MS (liquid chromatography / mass spectrometry) analysis was used according to conventional methods. A brief explanation is as follows. 10 ml of the culture was centrifuged (at 6 ° C for 5 minutes at 4 ° C for 5 minutes) to recover the cells. Cells were washed with 0.85% NaCl solution. The cells were then suspended in 200 μl of a 1: 1 mixed solution of acetonitrile and dimethylsulfoxide. Cell suspension was sonicated at 30 W for 20 minutes at 0 ° C using an ultrasonic shaker (UP200S sonicator, Hielscher Ultrasonic GmbH, Teltow, Germany) for 20 minutes to disrupt the cells. The cell lysate was centrifuged at 13,000 x g for 10 minutes at 4 ° C to obtain supernatant. The recovered supernatant was used as an LC / MS sample.

Example  5: Analysis results

The use of a medium with no added decanate and glycine resulted in no production of heme, but production of heme only when added with both a decanate and glycine. In addition to the addition of the peracinate and glycine, the addition of the pH buffering agent and bicarbonate increased the hem production by more than 60% as compared with the case of adding only the decanate and glycine. At this time, it was confirmed that maintaining the pH of the medium was a very important factor for efficient production of heme. In addition, the addition of growth factors had a positive effect on the production of heme. When compared with the case of addition of only the roticinate and glycine, the production of heme in the case of adding both of the pH buffer component, the bicarbonate and the growth factor was about 2 times Respectively. The result of the hem production confirmation is shown in FIG. It was also confirmed that supplying a mixed gas containing carbon dioxide instead of bicarbonate could provide a similar effect. Such a mixed gas supply method is considered to be more advantageous when the culture size is increased.

In addition, according to the present invention, it was confirmed that various porphyrin analogs were produced in addition to heme. The results confirmed by LC / MS are shown in Figs. 4 to 7. Fig. The LC / MS results were exactly the same as the theoretical estimates. The results of analysis of the production of various culture products including cell concentration change and heme according to the incubation time are shown in FIG.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (18)

A pharmaceutical composition comprising 5-aminolevir, which comprises bicarbonate as a saccharide, succinate, glycine, growth factor, and C4 pathway enhancer, wherein the concentration of bicarbonate is 0.01-50 g / (1-hydroxymethylbilane), uroporphyrin I, uroporphyrin III, coproporphyrin I, and the like. I), coproporphyrin III, protoporphyrin IX, or a biologically produced medium of heme. The culture medium according to claim 1, wherein the concentration of the rotinic acid is 0.01 to 50 g / L. The culture medium according to claim 1, wherein the concentration of the glycine is 0.01-20 g / L. The culture medium according to claim 1, wherein the growth factor is selected from thiamin, biotin, nicotinic acid, pantothenic acid, and cobalamin. The culture medium according to claim 1, wherein the concentration of the growth factor is 0.001-10 mg / L. The culture medium according to any one of claims 1 to 5, further comprising a pH stabilizing ability enhancing component, wherein the concentration of the pH stabilizing enhancing component is 10-500 mM. 7. The culture medium according to claim 6, wherein the pH-stabilizing ability enhancing component is phosphate. Preparing a medium as defined in claim 1; The prepared medium was supplemented with 5-aminolevulinic acid, porphyrin, porphobilinogen, 1-hydroxymethylbilane, uroporphyrin I, uroporphyrin III, Inoculating a production strain suitable for the biological production of coproporphyrin I, coproporphyrin III, protoporphyrin IX or heme; And a step of culturing a production strain. The method according to any one of claims 1 to 3, wherein the step (b) comprises culturing the strain a method for the biological production of uroporphyrin I, uroporphyrin III, coproporphyrin I, coproporphyrin III, protoporphyrin IX or heme. 9. The method according to claim 8, wherein the step of preparing the medium further comprises a pH stabilizing ability enhancing component in the medium, and the concentration of the pH stabilizing enhancing component is 10-500 mM. 10. The method of claim 9, wherein the pH stabilizing ability enhancing component is phosphate. Preparing a medium as defined in claim 1; The prepared medium was supplemented with 5-aminolevulinic acid, porphyrin, porphobilinogen, 1-hydroxymethylbilane, uroporphyrin I, uroporphyrin III, Inoculating a production strain suitable for the biological production of coproporphyrin I, coproporphyrin III, protoporphyrin IX or heme; A porphyrin, a porphobilinogen, a 1- (2-aminobenzoic acid), or a 5-aminolevulinic acid, which comprises culturing a production strain while supplying a mixed gas containing a carbon dioxide gas or a carbon dioxide gas. (1-hydroxymethylbilane), uroporphyrin I, uroporphyrin III, coproporphyrin I, coproporphyrin III, protoporphyrin IX ) Or a method of biological production of heme. 12. The method according to claim 11, wherein the step of preparing the medium further comprises a pH stabilizing ability enhancing component in the medium, and the concentration of the pH stabilizing enhancing component is 10-500 mM. 13. The method of claim 12, wherein the pH stabilizing ability enhancing component is phosphate. 12. The method according to claim 11, wherein the mixed gas contains carbon dioxide in an amount of 0.5-30%. delete delete delete delete

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