JP4935259B2 - Novel microorganism and method for producing carotenoids using the same - Google Patents

Description

  The present invention relates to a novel microorganism and a method for producing carotenoids, particularly canthaxanthin, using the same.

  Carotenoids such as astaxanthin, zeaxanthin, phenicoxanthin, canthaxanthin, adonixanthin, and β-carotene are added to foods, pharmaceuticals, quasi drugs, cosmetics and the like as natural pigments. Alternatively, the antioxidant function has attracted attention and is widely used as an antioxidant substance. These carotenoids are widely distributed in plants, animals, and microorganisms, and hundreds of types have been identified in nature (see, for example, Non-Patent Document 1).

  Bacteria belonging to the genus Agrobacterium (later reclassified to the genus Paracoccus) were reported by Yokoyama et al. As microorganisms that synthesize functional carotenoids (see, for example, Non-Patent Document 2). The bacterium is characterized by synthesizing a high content of astaxanthin, a functional carotenoid. In addition, the biosynthetic pathway of astaxanthin was elucidated in the same bacterium, and a detailed mechanism up to the gene level was reported (for example, see Non-Patent Document 3). According to the reported biosynthetic pathway, carotenoids passed through the metabolic synthesis pathway of the isoprenoid biosynthetic system starting from mevalonic acid, which is a basic metabolite, and the resulting 15-carbon farnesyl pyrophosphate (FPP) was further generated. By condensing farnesyl pyrophosphate with isopentenyl diphosphate having 5 carbon atoms (IPP), geranylgeranyl diphosphate having 20 carbon atoms (GGPP) is synthesized. Two molecules of geranylgeranyl diphosphate are then condensed to synthesize phytoene. Phytoene is converted to lycopene by a series of unsaturated reactions, and this lycopene is further synthesized into β-carotene by a cyclization reaction. Next, when β-carotene is oxidized by a ketolysis reaction, it becomes canthaxanthin, and when β-carotene is hydroxylated, it becomes zeaxanthin. Furthermore, the oxidation reaction by ketoization and hydroxylation proceeds, and astaxanthin is synthesized. The above carotenoid biosynthetic pathway is illustrated in FIG.

  On the other hand, it is reported that some microorganisms do not have a gene encoding the enzyme that ketolates the above-mentioned β-carotene and selectively synthesize only zeaxanthin (see, for example, Non-Patent Document 4). . Considering canthaxanthin in the same way, it is possible to selectively synthesize canthaxanthin if there is a microorganism lacking β-carotene hydroxylase, etc. Microbial separation has not been reported.

Canthaxanthin is useful as a functional carotenoid as a coloring agent for cultured fish, chicken eggs, etc., but it is only purified from a carotenoid extract and used. The carotenoid extract contains many carotenoids having chemical properties similar to those of canthaxanthin, and further makes extraction and purification difficult (see, for example, Patent Documents 1 and 2). Therefore, a substance such as a microorganism that accumulates canthaxanthin in a high content has been desired.
JP-A-6-237787 JP 2003-304875 A Eric A. Johnson et al. , Microbiological carotenoids, Advanced in Biochemical Engineering, Vol 53, p119-178 (1995). A. Yokoyama et al. , Production of Astaxanthin and 4-Ketzeaxanthin by marine bacteria, Agrobacterium aurantiacum, Biosci. Biotechnol. Biochem. 58: 1842-1844 (1994). N. Misawa et al. , Structure and functional analysis of a marine bacterial carotenoid biosynthesis biogenic synthesis and ataxanthin bioethy pathothepatho. Bacteriology, 177: 6575-6684 (1995). L. Pasamontes et al. , Isolation and charactorization of carotenoid biosynthesis genes of Flavobacterium sp. strain R1534, GENE, 185: 35-41 (1997).

  An object of the present invention is to provide a canthaxanthin selective synthetic bacterium which can easily extract and purify canthaxanthin which is a carotenoid. Another object of the present invention is to provide a method for producing canthaxanthin by a culture method.

  As a result of intensive studies to achieve the above-mentioned problems, the present inventors have found that canthaxanthin can be selectively synthesized by introducing mutations into a Paracoccus bacterium that produces carotenoids, leading to the present invention. That is, the present invention is a Paracoccus bacterium that selectively synthesizes canthaxanthin, which is obtained by breeding a carotenoid-producing Paracoccus bacterium. In addition, the present invention is a method for producing canthaxanthin characterized by culturing such bacteria and recovering canthaxanthin from the cells or culture solution. That is, the present invention relates to the following.

(1) carotenoid productivity Paracoccus bacteria TSAO538 strain (accession number FERM BP-10755).

(2) Carotenoids containing β-carotene, β-cryptoxanthin, echinenone, canthaxanthin, 3-hydroxyechinenone, 3′-hydroxyechinenone, zeaxanthin, phenicoxanthine, adonixanthin and astaxanthin produced (1) Paracoccus spp. TSAO538 strain, wherein canthaxanthin accounts for 90% by weight or more of the total amount of the carotenoids .

(3) A TSAO538 strain of the genus Paracoccus containing 1% by weight (in terms of dry weight) of canthaxanthin in the microbial cell .

(4) A method for producing a carotenoid, comprising culturing the Paracoccus genus TSAO538 strain according to any one of (1) to (3) and recovering carotenoids from the cultured cells or culture solution.

(5) The method for producing carotenoids according to (4) , wherein the carotenoid is canthaxanthin.

  The method for producing carotenoids of the present invention makes it possible to produce canthaxanthin useful as a food or feed on an industrial scale.

  Hereinafter, the present invention will be described in detail.

  The microorganism used in the present invention may be a wild strain or a mutant strain as long as it belongs to the genus Paracoccus and has the ability to synthesize canthaxanthin. Any strain can be used. Examples of the wild strain used in the present invention include Paracoccus sp. N81106 strain (patent biological deposit center accession number: FERM P-14023) can be mentioned. In the present invention, a microorganism that selectively synthesizes canthaxanthin by mutating a wild strain or a mutant strain that synthesizes carotenoids is more preferable.

  In the present invention, canthaxanthin synthesis ability can be imparted by introducing a mutation into a microorganism having astaxanthin synthesis ability. As shown in FIG. 1, in a microorganism having astaxanthin synthesis ability, β-carotene is hydroxylated and keto to synthesize astaxanthin as a final product. That is, in order to selectively synthesize canthaxanthin, a mutation may be introduced into crtZ, which is a gene encoding β-carotene hydroxylase, to reduce the hydroxylase activity. Alternatively, a mutation may be introduced into crtZ, which is a gene encoding β-carotene hydroxylase, and destroyed so that the gene does not function.

  In the present invention, the bacterium into which mutation is introduced is more preferably a bacterium having a carotenoid metabolic analog resistance having a high growth ability even if carotenoids are accumulated in cells at a high content. Examples of carotenoid metabolism analogs include α-ionone and β-ionone, which are well known to those skilled in the art. However, any carotenoid metabolism analog may be a substance that cancels the regulation of intracellular regulatory mechanisms by accumulation of carotenoids. For example, it is not limited to these substances. As used herein, “tolerance” means that the parent strain can grow even in the presence of a drug at a concentration that inhibits growth.

  Mutation treatment is a method well known to those skilled in the art for accelerating mutation by treating cells with mutagens or ultraviolet rays. Examples of mutagens include compounds such as N-methyl-N′-nitro-N-nitrosoguanidine and ethyl methanesulfonate.

  In the present invention, the β-carotene hydroxylase gene may be disrupted or deleted. Examples of methods for disruption and deletion include those in which those skilled in the art can insert appropriate DNA into a target gene by homologous recombination, which is one of well-known genetic engineering techniques. It is not limited to.

  In the present invention, after the mutation treatment, it is preferable to isolate the mutant strain and repeat breeding for evaluating a desired function. As an example of the breeding method, cells of Paracoccus genus strain TSTT052 (patent biological deposit center accession number FERM P-20690) obtained by culturing in advance were suspended in an aqueous solution of these mutagens and allowed to stand for a certain period of time. Later, the cells are collected by a method such as centrifugation to remove the mutagen. Thereafter, the cells are cultured on a plate medium and colonies of excellent strains are selected. Colonies are selected by selecting dark colonies, conducting liquid culture after separation, then extracting carotenoids from the cells and analyzing the amount and composition of carotenoids by HPLC etc. to narrow down the strains with improved productivity. Excellent strains can be obtained.

In the present invention, any microorganism may be used as long as it belongs to the genus Paracoccus and has the ability to synthesize canthaxanthin. For example, Paracoccus sp. There is TSAO538 strain (Accession No. FERM BP-10755 ).

This strain has the following mycological properties. In addition, + shows positive and-shows negative.
(A) Cell morphology Short bacillus 0.7-0.8 × 1.0-1.2 μm
(B) Spores −
(C) Motility −
(D) Colony morphology 1) Medium: Medium having the composition shown in Table 1 2) Diameter: 1.0 mm
3) Color tone: Orange 4) Raised state: Lens shape 5) Shape: Circular shape 6) Periphery: Full edge 7) Surface shape: Smooth 8) Transparency: Opaque 9) Viscosity: Butter-like
(E) Physiological properties 1) Catalase +
2) Oxidase −
3) Acid / gas generation from glucose-/-
4) O / F test on glucose-/-
(F) Usability of substrate 1) Methanol −
2) Ethanol-
3) Butanol +
4) Methylamine-
5) Glycerol-
6) Glucose +
7) Fructose +
8) Galactose +
9) Ribose-
10) Maltose +
11) Sucrose +
12) Lactose-
13) Mannitol +
14) Inositol-
15) Xylose-
16) Mannose-
17) Sorbitol +
18) Arabinose-
19) Trehalose +
20) Gluconic acid-
21) Acetic acid-
22) Lactic acid +
23) Citric acid-
24) Succinic acid + W
25) Malonic acid + W
26) Pyruvate + W
27) Trimethyl-N-oxide-
28) Thiosulfuric acid −

  In the present invention, the selective synthesis means that canthaxanthin can be synthesized in a microorganism that synthesizes carotenoids so that canthaxanthin can be easily separated and purified. -Carotenoids containing carotene, β-cryptoxanthin, echinone, canthaxanthin, 3-hydroxyechinenone, 3'-hydroxyechinenone, zeaxanthin, phenicoxanthine, adonixanthin and astaxanthin, where canthaxanthin is a carotenoid 90% by weight or more of the total amount. The carotenoids include β-carotene, β-cryptoxanthin, echinone, canthaxanthin, 3-hydroxyechinenone, 3′-hydroxyechinenone, zeaxanthin, phenicoxanthine, adonixanthin, astaxanthin, and the like. And other carotenoids that accumulate in the microorganism.

  In the present invention, an excellent strain can be determined by evaluating an appropriate strain. When a solid medium is used, it is possible to evaluate by picking up an arbitrary colony, performing liquid culture, and evaluating the growth ability and carotenoid production ability.

  The microorganism used in the method of the present invention can be cultured in a nutrient medium known as a conventional method. As the medium used in the present invention, any medium can be used as long as bacteria can grow and produce carotenoids. The carbon source can be molasses, glucose, fructose, maltose, sucrose, starch, Lactose, glycerol, acetic acid, etc., nitrogen sources include natural ingredients such as corn steep liquor, peptone, yeast extract, meat extract, soybean meal, ammonium salts such as ammonium acetate, ammonium chloride, ammonium sulfate, glutamic acid, aspartic acid, Amino acids such as glycine, inorganic salts include phosphates such as monosodium phosphate, disodium phosphate, monopotassium phosphate, dipotassium phosphate, and sodium chloride, and metal ions include magnesium chloride and magnesium sulfate. , Ferrous sulfate, ferric sulfate, ferrous chloride, ferric chloride, iron citrate, sulfuric acid Numonium iron, calcium chloride dihydrate, calcium sulfate, zinc sulfate, zinc chloride, copper sulfate, copper chloride, manganese sulfate, manganese chloride, etc. are yeast extracts, biotin, nicotinic acid, thiamine, riboflavin, inositol as vitamins , Pyridoxine and the like can be used.

  The strain used in the culturing step can be transferred from the streak culture plate to the fermentation vessel according to a known method. A preferred method is a method using an agar plate medium, a slant agar medium and a flask culture solution.

  The conditions for culturing a novel microorganism under conditions for producing a carotenoid such as canthaxanthin of the present invention may be carried out using any general method. In a preferred embodiment of this method, the culture is preferably performed in a culture solution. Regarding this submerged culture, the conditions used in normal submerged culture may be used. Preferably, the culture temperature is set to 10 to 35 ° C., the pH of the medium is set to a range of 6 to 9, and fermentation is performed for 20 to 200 hours. Regarding the culture temperature, the temperature may be changed at each stage by distinguishing between the initial stage, the middle stage, and the late stage. Suitable conditions using the nutrient medium of the present invention are a culture temperature of 20 to 27 ° C., a pH of about 7.0, and a culture time of 50 to 150 hours.

  The pH of the medium is adjusted to 6.5 to 8.0, preferably about 7.0. The pH is adjusted using an aqueous solution of sodium hydroxide, potassium hydroxide or ammonia, but is not limited to these.

  The method for analyzing carotenoids in the present invention is not particularly limited as long as it is stably and efficiently recovered from the cells or the culture solution. For example, the extraction solvent is methanol, ethanol, isopropyl alcohol, acetone, methyl ethyl ketone, methyl isobutyl ketone, dichloromethane. , Chloroform, dimethylformamide, dimethylsulfoxide and the like are preferable. The quantification of the extracted carotenoid is preferably performed by high performance liquid chromatography in which various carotenoids are separated and excellent in quantification.

  In order to recover carotenoids and / or canthaxanthin from the microbial cells or the culture solution after the culture, the microbial cells may be separated from the culture solution by, for example, centrifugation and extracted from each with an appropriate organic solvent. Examples of the organic solvent include methanol, ethanol, isopropyl alcohol, acetone, methyl ethyl ketone, methyl isobutyl ketone, dichloromethane, chloroform, dimethylformamide, dimethyl sulfoxide and the like. A preferable example is acetone. Furthermore, it can be separated and purified with high purity using liquid chromatography or the like. Examples of the separation principle of liquid chromatography include ion exchange, hydrophobic interaction, molecular sieving and the like. Preferred are reverse phase chromatography and normal phase chromatography. Alternatively, it is extracted from the cells by supercritical fluid extraction.

  Or after completion | finish of culture | cultivation, you may isolate | separate a microbial cell from a culture medium using methods, such as centrifugation operation, decantation, or filtration. The obtained bacterial cells are made into a slurry by adding water to a viscosity that is easy to use. In order to prevent decomposition of carotenoids such as canthaxanthin, an appropriate additive may be added to the slurry. Examples of the additive include antioxidants such as ascorbic acid, but are not limited thereto. Thereafter, the prepared slurry is homogenized using, for example, a crusher using glass beads or zirconia beads or a high-pressure homogenizer, and dried for later use. A preferred drying method is spray drying.

  You may add this microbial cell as it is, for example in feeds, such as cultured fish. Alternatively, as described above, it may be extracted with a polar organic solvent or the like. The cell body, which contains little pigment and remains after extraction of carotenoids such as canthaxanthin, can be used as an ideal source of protein and vitamins for raising poultry.

  Hereinafter, although it demonstrates still in detail using an Example, this invention is not limited to these.

Example 1
Mutagenic introduction into Paracoccus bacterium and plate evaluation of the mutant strain Paracoccus bacterium TSTT052 strain (Accession No. FERM P-20690) was inoculated into 5 ml of the liquid medium shown in Table 1, and cultured with shaking at 25 ° C. and 150 rpm for 1 day. went. 1 ml of this culture solution was transferred to a 1.5 ml Eppendorf tube, and the cells were collected by centrifugation at 15,000 rpm for 10 minutes. This microbial cell was suspended in 1 ml of 0.1 M potassium phosphate buffer (buffer A) at pH 7.0, and then 3 mg / ml N-methyl-N′-nitro-N-nitrosoguanidine (hereinafter referred to as NTG). (Abbreviated) 10 μl of aqueous solution was added and allowed to stand for 30-60 minutes. Thereafter, the supernatant was removed by centrifugation, and the operation of resuspending in buffer A was repeated twice to remove NTG. Further, the cells were suspended in 0.5 ml buffer A, inoculated into 3 ml of the medium shown in Table 1, and cultured for 4 to 5 hours. An appropriate amount of the obtained culture broth was spread on a plate obtained by further adding agar to a medium having the composition shown in Table 1 and solidified, followed by culturing at 25 ° C. for 5 days. From the third day of the culture, the colonies could be visually confirmed, and the red shades of the colonies could be distinguished.

(Example 2)
Culture of Paracoccus spp. Bacterial strains The strains obtained in Example 1 were randomly selected, inoculated into 5 ml of the culture solution shown in Table 1, and cultured at 25 ° C. for 1 day. Next, 60 ml of the medium having the composition shown in Table 1 was placed in a 100 ml baffled Erlenmeyer flask and sterilized, and 2 ml of the previously cultured medium was inoculated. After culturing at 25 ° C. for 7 days (100 rpm), the culture solution was appropriately removed, and turbidity (OD660 nm) and carotenoid were quantified. Carotenoids were quantified as follows. First, 1 ml of the culture solution was placed in a 1.5 ml Eppendorf tube and centrifuged at 15,000 rpm for 5 minutes to obtain a cell pellet. The cells were suspended in 20 μl of pure water, and then 200 μl of dimethylformamide and 500 μl of acetone were added and shaken to extract carotenoids. After the residue was removed by centrifugation at 15,000 rpm for 5 minutes, various carotenoids were quantified by high performance liquid chromatography (hereinafter abbreviated as HPLC) using a TSKgel-ODS80TM column (manufactured by Tosoh Corporation). Carotenoids were separated using a 5:95 mixed solvent of pure water and methanol as the A liquid, and a 7: 3 mixed solvent of methanol and tetrahydrofuran as the B liquid, and the A liquid was passed through the column for 5 minutes at a flow rate of 1 ml / min. Then, a linear concentration gradient of 5 minutes was performed from the same flow rate A liquid to B liquid, and B liquid was further passed for 5 minutes. The canthaxanthin concentration was determined by monitoring the absorbance at 470 nm and calculating the concentration from a calibration curve prepared with a known concentration of canthaxanthin standard reagent. Among the evaluated mutant strains, TSAO538 strain (a strain deposited at the Patent Organism Depositary, accession number FERM BP-10755 ) was isolated as a new mutant strain that could confirm canthaxanthin selective synthesis. Canthaxanthin was confirmed from the retention time on HPLC of the standard reagent. FIG. 2 shows the HPLC pattern of the extracted carotenoid of TSAO538 strain and that of the standard reagent. As shown in FIG. 2, the extracted carotenoid of the TSAO538 strain overlapped with the peak of the standard reagent canthaxanthin, and it was confirmed that canthaxanthin was selectively synthesized.

  The production pattern of carotenoids on the fourth day of culture of the TSTT052 strain, which is the same strain cultured in the same manner, is shown in FIG. As shown in FIG. 2 and FIG. 3, the TSAO538 strain, unlike the TSTT052 strain, selectively synthesized canthaxanthin, and the proportion of canthaxanthin in carotenoids exceeded 90%. The selective synthesis ratio is determined by HPLC method (for example, see Non-patent Document 2), and the production amount of astaxanthin, zeaxanthin, canthaxanthin, β-carotene, phenicoxanthine, adonixanthin, and echinenone is obtained, and production of canthaxanthin relative thereto is obtained. Calculated as a quantity. The results are shown in Table 2.

  FIG. 4 shows a graph showing the change over time in the synthesis of canthaxanthin of the TSAO538 strain. As shown in FIG. 4, the TSAO538 strain synthesized canthaxanthin as the culture progressed, and contained canthaxanthin in the cells without degradation even on the fifth day of culture. On the fourth day of culture, the canthaxanthin content per cell was about 1.2% by weight. The weight% was calculated by determining the weight of the dry cells.

  The novel canthaxanthin selective synthetic bacterium of the present invention can easily extract and purify canthaxanthin, which is a carotenoid, so that canthaxanthin useful as food or feed can be produced on an industrial scale.

It is a pathway diagram starting from phytoene, chemically modifying β-carotene, and finally producing astaxanthin. It is a HPLC chart of carotenoids extracted from TSAO538 strain, in which X-axis (horizontal axis) indicates retention time (unit: minutes), Y-axis (vertical axis) HPLC peak intensity (unit: mV (arbitrary intensity)) ). It is a HPLC chart of carotenoids extracted from the TSTT052 strain, in which X-axis (horizontal axis) indicates retention time (unit is minutes), Y-axis (vertical axis) HPLC peak intensity (unit is mV (arbitrary intensity)) ). It is a figure which shows the culture | cultivation progress of TSAO538 strain | stump | stock, in which X-axis (horizontal axis) shows the number of culture days (unit is a day), Y-axis (left vertical axis) is OD660nm absorbance value, Y-axis (right vertical axis) ) Indicates the carotenoid concentration (unit: mg / L).

Explanation of symbols

1: Absorbance value at OD 660 nm as the culture progresses (▲)
2: Total carotenoid concentration during the course of culture (●)
3: Concentration of canthaxanthin (○)

Claims (5)

Carotenoid productivity Paracoccus bacteria TSAO538 strain (accession number FERM BP-10755). In carotenoids containing β-carotene, β-cryptoxanthin, echinenone, canthaxanthin, 3-hydroxyechinenone, 3′-hydroxyechinenone, zeaxanthin, phenicoxanthine, adonixanthin and astaxanthin to be produced, The Paracoccus bacterium TSAO538 strain according to claim 1, wherein xanthine accounts for 90% by weight or more of the total amount of the carotenoids. The Paracoccus bacterium TSAO538 strain according to claim 1, wherein the fungus body contains canthaxanthin in an amount of 1% by weight (in terms of dry weight) or more. A method for producing carotenoids, comprising culturing the Paracoccus bacterium TSAO538 strain according to any one of claims 1 to 3 and recovering the carotenoids from the cultured cells or culture solution. The carotenoid production method according to claim 4 , wherein the carotenoid is canthaxanthin.

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