KR19990048085A - Astaxanthin-producing yeast mutants and methods for their production - Google Patents

Abstract

The present invention relates to a mutant strain of Phaffia rhodozyma which is highly efficient in producing astaxanthin pigment which is a carotenoid-based pigment. The present invention relates to a mutant strain of Papia rhodozyma mutants KT-9 and KT-12 Has total carotenoid productivity of about 410,000 g / l and 620,000 g / l.

Description

Astaxanthin-producing yeast mutants and methods for their production

The present invention relates to a Phaffia rhodozyma mutant having high astaxanthin productivity and a process for producing the same.

Astaxanthin (3,3'-dihydroxy-β, β'-carotene-4,4'-dione) is a carotenoid pigment having the following formula:

The astaxanthin pigment is deposited in animal blood vessels, accumulated in adipose tissue, and finally accumulated in target tissues. The color of the shell of shrimp and crawfish, the beak and yolk of birds, and the color of fish such as salmon and trout To pink, resulting in a distinctive nutrient and flavor effect as well as color enhancement. In addition, astaxanthin has excellent properties such as color tone and thermal stability, and has been proved to exhibit superior antioxidative effects to other carotenoids and tocopherol. Thus, astaxanthin has attracted considerable attention not only in the food industry but also in the pharmaceutical field as an antioxidant pigment (Kurashige, M., et al, Physiol Chem Phys Med NMR 27-38 (1990);...... Terao, Lipids 24, 659 (1989);.. Miki, W. Pure Appl Chem 63, 141-146 (1991)). In particular, it exhibits an important metabolic role in humans due to the precursor of vitamin A, the activation of immune function, and the anticancer action by the removal of oxygen radical (Goodwin, TW Metabolism, Nutrition and Function of Carotenoids. Ann. Rev. Nutr . (1986), Benidch, A., et al., Biological actions of carotenoids, FASEB J. 3 , 1927 (1989)).

Consumption of seafood continues to increase because modern diets tend to avoid animal fat and cholesterol-containing foods, and about 15% of the consumed seafood is produced in aquaculture. Among these fish, salmon is one of the important aquaculture resources, and since it can not synthesize its own pigment, it has to be fed from the outside as feed. As a feed source, yeast Papi Rhodozima, which contains astaxanthin pigment, is widely used as a nutrient and pigment source of fish. As a result, the amount of astaxanthin pigment is increasing worldwide, and accordingly, in order to smoothly supply the pigment to the market, a large amount of pigment has been produced by a chemical synthesis method rather than a natural extraction method of natural pigment. However, synthetic pigments have not been recognized by the US Food and Drug Administration (FDA) because their bioavailability is low and their stability is not ensured.

Accordingly, researches have been conducted to mass-produce pigments by culturing microorganisms capable of producing astaxanthin, which is a natural pigment. However, wild type yeast Paipi Rhodozima has a limited production amount of natural astaxanthin and thus has limited industrial use. Therefore, studies for improving the production ability of natural astaxanthin pigment by developing a mutant strain of yeast Paipi Rhodozima by strain improvement are actively being actively carried out.

As for the microorganisms producing astaxanthin pigment, in 1972, Herman Papp et al. Found papi rhodozima in Japan, Alaska and the former Soviet Union (Phaff et al., A comparative study of the yeast flora associated with trees on the In 1976, Miller et al . (1986) reported that P. aeruginosa, which reproduces in cold areas such as mountainous areas, is a carotenoid It is reported to be a yeast that produces astaxanthin, a kind of pigment. In addition, Andrew et al. Have found that this yeast produces echinenone, hydroxyechinenone, and phenicoxanthin as intermediates in the synthesis of astaxanthin as carotenoids other than astaxanthin (Fig. 1), but it has been reported that about 85% or more of the total amount of these carotenoids produced in Papia rhodozyma is astaxanthin (Andrews, AG, et al., Carotenoids of Phaffia rhodozyma, a Red- Pigmented Fermenting Yeast, Phytochemistry, 15 , 1003-1007 (1976)).

It is also known that marine microorganisms and freshwater organisms can produce astaxanthin (3,3'-dihydroxy-4,4'-diketo-β-carotene). It is known that Hamatococcus pluvialis , a kind of algae, has a high ability to produce astaxanthin pigment, and their ability to produce astaxanthin can be improved by improving the culture conditions of these strains. Research is also underway to improve. However, this strain has been limited in use since it has to be cultured in ponds for a long period of time and the pigment must be extracted only from the cells after production (Droop, MR Carotenogenesis in Haematococcus pluisalis. Nature 175 , 42 (1955); Carotenoid synthesis in the alga Haematococcus pluvialis, J. Biochem. 57 , 376 (1954)).

In addition there are ahseuta Santin marine microorganism Agrobacterium William (Agrobacterim) as production strain, is currently extracted a gene from here, rather than how to produce the pigment from the microbe itself has made an attempt to produce the pigment from the yeast. However, this method also makes it difficult to increase the production efficiency by introducing 6 or more genes into baker 's yeast. The reason for this is that the production of astaxanthin is understood to be a consequence of many genes involved in pigment production besides the structural gene of carotenoid (Yamano, S. et al., Metabolic engineering for production of β-carotene and lycopene on Sacharomyces cerevisiae. Biosci. Biotech. Biochem., 58 , 112 (1994)).

The characteristic of yeast Papi Rhodozima is that it is a single cell microorganism that reproduces sexually, and most of its life forms a colony with a pink pigment in spherical or oval shape. As an aerobic bacteria, it does not ferment alcohol, but produces astaxanthin, which has a powerful antioxidant effect by protecting against oxygen radicals.

On the other hand, since the yeast Papi Rhodozima grow at a lower temperature than other yeast, it requires a long period of cultivation for about 5 to 6 days, and accordingly, a lot of cost is consumed, and a large amount of astaxanthin It accumulates after stopping. Therefore, proper cooling and cooling of the cold air and the aeration of the air are necessary conditions, and accordingly, research on cost reduction is urgently required.

In addition, the natural Papi Rhodozima has a considerable limitation in commercial use due to low natural astaxanthin pigment productivity. In order to be able to compete with the synthetic astaxanthin pigment, at least 6 mg of astaxanthin per g of yeast can be mass produced The mutant strains need to be improved.

The present inventors have already developed mutant HP-30, R10-1, p1-2 and M-18 of Papiarodojima, which has already produced a large amount of astaxanthin pigment, and have proposed Korean Patent Application No. 95-4260 34396). The present inventors completed the present invention by continuing the study on the HP-30 mutant strain and developing a mutant strain of yeast Papiarodojima whose production amount of pigment is further increased.

Accordingly, an object of the present invention is to provide a mutant strain which mass-produces a natural pigment astaxanthin.

Another object of the present invention is to provide a method for producing said mutant strain.

Figure 1 shows the synthesis step of astaxanthin and its intermediates.

Fig. 2 is a schematic diagram showing the production process of Papia rhodozima KT-9 and KT-12.

3 is a photomicrograph (1,500 times magnification) of Papia Rhodozima KT-9.

4 is a photomicrograph (1,500 times magnification) of Papia rhodozima KT-12.

According to the above object, in the present invention, a mutant strain KT-9 and KT-12 having high productivity of astaxanthin are provided.

According to another object, in the present invention, gamma ray irradiation, N-methyl-N-nitro-N-nitrosoguanidine (NTG) treatment and thymol treatment are applied to P. pneumoniae strains, To 99.9% of the mutant strain KT-9, respectively, in any order.

In the present invention, NTG treatment, beta-ionone treatment, triethylamine (TEA) treatment, and beta-carotene treatment were applied to P. typhimurium KT-9 strains at an extinction ratio of 60 To 99.9% of the mutant strain KT-12, respectively, in any order.

Hereinafter, the present invention will be described in detail.

As used herein, the term 'pigment productivity' refers to the amount (ig) of pigment obtained per liter of culture. The yield (Yp / x) of the total carotenoid production represents the yield of carotenoid with respect to the amount of the cell mass, wherein x is the amount of the cell mass s is the cell mass, s is the substrate, Y is the yield, and p is the product carotenoid.

The improved yeast strains of the present invention can be produced by the following method.

(Deposited with the Agricultural Research Culture Collection (NRRL) as NRRL Y-21359, dated December 2, 1994) was suspended in YM liquid medium (0.3% yeast The yeast was killed by centrifuging the suspension in 0.3% malt extract, 0.5% peptone, and 1.0% glucose, suspended in a buffer, and irradiated with a gamma ray to induce physical mutagenesis. YM liquid culture medium, 0.7-3.0 KGy, preferably 2.6 KGy, such that the mutant yeast cells kill 60% to 99.9%, preferably 95% to 99.9% of gamma-rays emitted from [ ⁶⁰ Co] To the cell suspension.

The suspension was inoculated in a YM liquid medium and cultured. The culture was diluted in YD solid medium (0.5% yeast extract, 0.6 M potassium chloride, 1.0% glucose, 1.8% agar) and cultured. The resulting colonies were dark pink Of the colonies. The selection procedure on the YD solid medium is repeated 2 to 5 times to obtain pure colonies. After culturing these strains in YM liquid medium for 5 days, the strain yield and total carotenoid yield were measured and compared, and one strain with the highest pigment yield was selected. This strain is fed-fed and more accurately measures cell yield and total carotenoid production yield. The gamma-ray irradiation and sorting process is repeated one to five times, preferably twice.

The selected mutant yeast is cultured in YM liquid medium and treated with NTG to kill the yeast. At this time, depending on the concentration of NTG, the treatment temperature, and the treatment time, the yeast kill rate may vary, and the probability of obtaining the mutant varies depending on the kill rate. Therefore, these conditions are appropriately controlled, and the kill rate is 60% to 99.9% To 95% to 99.9%. The surviving yeast was diluted in YM solid medium and cultured. The resulting colonies were selected for all of the strains which are faster than the parent strain and have a shiny and pinkish color. The selection procedure on the YD solid medium is repeated 2 to 5 times to obtain pure colonies. After culturing these strains in YM liquid medium for 5 days, the strain yield and total carotenoid yield were measured and compared, and one strain with the highest pigment yield was selected. This strain is fed-fed and more accurately measures cell yield and total carotenoid production yield. The NTG treatment and screening process is repeated 1 to 5 times, preferably once.

The mutant yeast by NTG is then cultured in YM liquid medium and then killed by diluting it in a semi-molten YM solid medium which is a chemical inhibitor. At this time, depending on the concentration of the cymol, the treatment temperature and the treatment time, the killing rate of the yeast may vary, and the probability of obtaining the mutant varies depending on the killing rate. Therefore, the killing rate is 60 to 99.9% Respectively. Preferably 80 to 90%, more preferably 85%. Yeast survived in the cymolitic YM solid medium was compared with the parent strain to select all the strains showing rapid growth and dense pink colonies. The selection procedure on the YD solid medium is repeated 2 to 5 times to obtain pure colonies. After culturing these strains in YM liquid medium for 5 days, the strain yield and total carotenoid yield were measured and compared, and one strain with the highest pigment yield was selected. This strain is fed-fed and more accurately measures cell yield and total carotenoid production yield. The above-mentioned process for the silymal treatment and the screening is carried out once to 5 times, preferably once, repeatedly.

The order of the above gamma ray irradiation, NTG treatment, and the silyl treatment may be arbitrarily changed, and it is preferable that the irradiation is performed in the order of 2 times of gamma ray irradiation, NTG treatment, and silyl treatment as shown in FIG. 2 .

The selected strains were named as Papi Rhodozima KT-9 and deposited at Korean Culture Center of Microorganism (KCCM) as the deposit number KCCM-10112 dated October 7, 1997.

The mutant strain KT-9 is cultured in a YM liquid medium, and NTG treatment and screening are again carried out in the same manner as described above. The NTG selection process is performed once to five times, preferably three times.

The mutant strain selected by NTG treatment is cultured in YM liquid medium and then killed by dilution in YM solid medium treated with beta-ionone, a chemical inhibitor. The beta-ionone treatment and screening process is similar to that of the above-mentioned cymol except that the rate of death is controlled to be 60 to 99.9%, preferably 70 to 80%, more preferably 75%. The same method is used.

The selected mutant strain is cultured in a YM liquid medium and then killed by diluting it in a YM solid medium treated with a chemical inhibitor, triethylamine. At this time, the treatment and selection of triethylamine are carried out in the same manner as in the case of the above-mentioned cymol.

The selected mutant strain is cultured in YM liquid medium and then killed by diluting it in YM solid medium treated with beta-carotene, which is a chemical inhibitor. The treatment and screening of beta-carotene is carried out in the case of the above-mentioned cymol, except that the rate of killing is controlled to be 60 to 99.9%, preferably 90 to 99.9%, more preferably 95% The same method is used.

The order of NTG treatment, beta-ionone treatment, triethylamine treatment and beta-carotene treatment may be arbitrarily changed, and preferably NTG treatment, beta-ionone treatment, NTG Treatment, triethylamine treatment, NTG treatment and beta-carotene treatment in that order.

The selected strains were named as Papi Rhodozima KT-12 and deposited as KCCM-10113 deposited on October 7, 1997 in the Korean Society for Microorganism Conservation.

The method of fed-batch culture of the strain is as follows.

First, the mutant strain of P. typhimurium is shake cultured in a YM liquid medium at 18-22 DEG C for 5 days to activate the microorganism, and then cultured in an initiation medium containing 5 ppm or more of biotin and 0.05% or more of yeast extract, Incubate for 6 days. For fed-batch culture using an agitation type fermenter, the above-prepared seeds are inoculated in a fermenter containing the starting medium of the same composition as above to pH 5.0 to 7.0, preferably pH 6.0, stirring speed 200 to 600 rpm, Is cultivated for 5 to 8 days while maintaining the culture at 300 rpm and culturing at 18 to 23 캜, preferably 21 캜, aeration amount of 0.5 to 1.5 vvm, preferably 1 vvm, and when the sugar in the culture medium is completely consumed, Or more of the carbon source is supplied so that the sugar concentration is maintained at 1% or less while the fed-batch culture is carried out. As a starting medium used for the above-mentioned fed-batch culture, 40.0 g of glucose, 7.0 g of peptone, 5.0 g of CSL, 5.0 g of yeast extract, 4.0 g of malt extract, 2.5 g of ammonium sulfate, 1.0 g of ammonium acetate, A medium containing 1.0 g of potassium phosphate, 0.8 g of dibasic ammonium phosphate, 0.5 g of magnesium sulfate, 3.0 ml of silicone oil, 10.0 mg of thiamine, 6.0 mg of copper sulfate, 5.0 mg of biotin, 5.0 mg of iron chloride and 4.0 mg of manganese sulfate As the feed medium, a medium containing 450.0 g of glucose per liter of medium and 5.0 g of ammonium sulfate is preferable.

The method of measuring the dry cell mass is as follows.

The Papi rhodozyma mutant strain is cultured in YM liquid medium for 5 days or the cultured liquid is cultured at 70 to 120 DEG C, preferably 100 DEG C for 5 to 25 minutes, preferably 15 minutes After the heat treatment, the cells are recovered by centrifuging 5 ml of the culture solution at 6,000 to 10,000 rpm, preferably 9,000 rpm for 5 to 15 minutes, preferably 10 minutes, washed twice with distilled water, Dry for a period of time and measure the cell dry weight.

As a method for extracting and quantifying the total carotenoid, various methods known in the art can be used from the strain. For example, the cell wall is disrupted with dimethylsulfoxide (DMSO) and the amount of the extracted pigment is quantified . Namely, in order to measure the total amount of the pigment contained in the cells, 1 ml of the cell culture solution in which the above-mentioned fed-batch culture was completed was put in a centrifuge tube, 9 ml of distilled water was added, and then centrifuged at 9,000 rpm for 10 minutes to wash the precipitate, To remove water. 5 to 20 ml, preferably 10 ml of a pre-warmed dimethylsulfoxide stock solution at 35 to 65 ° C, preferably 55 ° C, is added and reacted at the same temperature for 5 to 10 minutes, preferably 8 minutes, Allow yeast cells to disintegrate sufficiently. 3 ml of a 1.0 M phosphate buffer solution (pH 7.0) and 10 to 30 ml of a solvent mixed with an equal amount of hexane and ethyl acetate are added to the cell lysate so that the pigment is well extracted in the solvent layer, preferably 20 Ml, and the mixture was centrifuged to remove impurities in the solvent layer, and the absorbance of the supernatant was measured at 474 nm to quantify the total pigment contained in the cells.

Total carotenoids can be quantitated by the following equation by Johnson et al . Using the 1% extinction coefficient (2,100) from the absorbance and the cell weight obtained above (Johnson, EA et al ., Isolation of Phaffia rhodozyma mutants with increase astaxanthin content, Appl. and Environ. Microbiol., 55 , 116-124 (1989)).

Total carotenoid amount (/ / g yeast dry weight) = (S x A ₄₇₄ x 100) (21 x D)

Where S is the amount of solvent used in the extraction (ml); A ₄₇₄ is the absorbance of the dye measured at ₄₇₄ nm; D is the dry weight of the yeast used.

In the above equation, the dry weight of the yeast used for pigment analysis should be measured to determine the total amount of carotenoid. However, since the yeast used for pigment analysis is too small to directly measure the dry weight, 10 times the amount of yeast culture used is washed with water, dried at 105 ° C until there is no weight change, We divide it.

In the above pigment extraction and quantification method, changes in the total amount of carotenoids and the rate of strain destruction can be observed by changing various conditions such as culture time and treatment time even in the same strain.

The strain KT-9 of the present invention has the form of FIG. 3 and has a total yield (Yp / x) of carotenoid including astaxanthin at a yield of 63 g / L (Yx / s) and 6,573 g / Has a pigment productivity of 414,099 占 퐂 / l. The strain KT-12 has the form of FIG. 4 and has a total yield (Yp / x) of the carotenoid including astaxanthin at a yield of 73 g / l (Yx / s) and 8.434 ug / g, Lt; / RTI > of pigment productivity.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Reference Example 1: Fed-batch culture

The fed-batch culture in the examples was carried out as follows.

First, the papi rhodozyma mutant yeast strain was inoculated into a 250 ml Erlenmeyer flask containing 30 ml of YM medium and cultured at 21 DEG C with stirring at 150 rpm for 4 days. Then, the culture medium was added to the starting medium (40.0 g glucose per liter, peptone 7.0 5.0 g of CSL, 5.0 g of malt extract, 4.0 g of malt extract, 2.5 g of ammonium sulfate, 1.0 g of ammonium acetate, 1.0 g of potassium phosphate, 0.8 g of dibasic ammonium phosphate, 0.5 g of magnesium sulfate, , 10.0 mg of thiamine, 6.0 mg of copper sulfate, 5.0 mg of biotin, 5.0 mg of iron chloride and 4.0 mg of manganese sulfate) was inoculated in a 1 L Erlenmeyer flask and cultured under the same conditions as above for 3 days.

The culture was inoculated in a 5 L fermenter containing 1.8 L of the starting medium and cultured at a stirring speed of 300 rpm, a culture temperature of 21 DEG C, aeration amount of 1 vvm, and a pH of 6.0. After the sugar was depleted, And 5.0 g of ammonium sulfate) were cultured for 6 days while being supplied to a metering pump so that the sugar concentration in the fermenter was kept at 1% or less.

Reference Example 2: Measurement of dry cell weight

The measurement of the dry cell mass in the examples was carried out in the following manner. That is, the colonies were cultured for 5 days in YM liquid medium or cultured in the same manner as in Reference Example 1. After the cultivation was completed, the culture medium was heat-treated at 100 ° C for 15 minutes, and 5 ml of the heat-treated culture was centrifuged at 9,000 rpm for 10 minutes The cells were recovered, washed twice with distilled water, dried at 105 DEG C for about 15 hours, and the dry weight was measured.

Reference Example 3: Quantification of total carotenoids

Quantitative determination of the pigment in the examples was carried out in the following manner. That is, the colonies were cultured for 5 days in YM liquid medium or cultured in the same manner as in Reference Example 1. After the cultivation was completed, 1 ml of the culture broth was placed in a 50 ml centrifuge tube, 9 ml of distilled water was added, Was washed twice with distilled water and water was removed using a vacuum dryer. Then, 10 ml of dimethylsulfoxide preheated at 55 ° C was added thereto, reacted at the same temperature for 5 minutes, and then shaken vigorously to disrupt the cells. 3 ml of 1.0 M phosphate buffer solution (pH 7.0) was added to the cell lysate, and 20 ml of an equal volume of a solvent mixture of hexane and ethyl acetate was added to extract the carotenoid pigment as a solvent layer. The solvent layer was centrifuged at 9,000 rpm for 5 minutes to remove polymer impurities, and the absorbance of the supernatant was measured at 474 nm to quantify total carotenoids contained in the cells.

Comparative Example: Pigment yield of Papi Rhodozima HP-30 strain

Papier Rhodozima HP-30 (NRRL Y-21359) was prepared on a slant medium prepared using sterile YM solid medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1.0% glucose, 1.8% agar) The cells were subcultured every week and used for experiments while refrigerated.

The Papia rhodozyma HP-30 strain was cultured in the same manner as in Reference Example 1, and the dried cell mass and the total carotenoid were measured in the same manner as in Reference Examples 2 and 3. As a result, the HP-30 strain of Paia rhodozima has a total carninoid production yield (Yp / x) of 4,200 ㎍ / g and a bacterial yield (Yx / s) of 60 g / ℓ and overall productivity of 252,000 ㎍ / ℓ And it was found.

Example 1: Selection of Papiarodojima strain KT-9

(Step 1) inducing primary mutation by gamma irradiation

After the medium was inoculated into a YM liquid medium and shaken at a rotation speed of 150 rpm for 5 days while keeping 21 ° C, 5 ml of this culture was added to a YM liquid medium 45 (NRRL Y-21359) having the same composition Ml and cultured further under the same conditions for 3 days. Thereafter, the culture was centrifuged using a sterilized centrifuge tube, washed with 0.1 M phosphate buffer solution (pH 7.0), suspended in the same buffer to an absorbance value of 2.3 at 600 nm, Were used for mutation by irradiation.

As a method of inducing mutation by irradiation with gamma rays, gamma ray emitted from [ ⁶⁰ Co] was irradiated to the cell suspension at 2.6 KGy, in which at least 90% of the cells were dead, and the suspension was put in a YM liquid medium and cultured for about 3 days This culture was diluted in YM solid medium and cultured at 21 ° C for 10 days. At this time, the culture solution not irradiated with gamma ray was diluted and cultured in YM agar medium in the same manner, and the fungicidal rates of the gamma-ray treated group and the non-treated group were compared. The resulting colonies were plated on YD solid medium (0.5% yeast extract, 0.6 M potassium chloride, 1.0% glucose, 1.8% agar) as a selection medium and incubated for 10 days at 21 ° C Lt; / RTI > The yields of the colonies were slightly lowered, but all of the dark pink colonies which were excellent in pigment formation, glossy, and stable were selected. Stability indicates that colonies are uniformly formed when cultured in a flat plate. The selection procedure in the YD solid medium was repeated three times to obtain pure colonies.

The selected colonies were cultured in each YM liquid medium for 5 days, and then the dried cell mass and the total carotenoid were measured in the same manner as in Reference Examples 2 and 3 to select one strain having the highest pigment yield.

One selected strain was cultivated in the same manner as in Reference Example 1 and cultured in a fed-batch manner. The dried cell mass and the total carotenoid were measured in the same manner as in Reference Examples 2 and 3, and the selected strain had a cell yield Yx / s) and the total carotenoid production yield (Yp / x) including astaxanthin pigment was 4,925 ㎍ / g, indicating that the total pigment yield was 285,650 ㎍ / ℓ.

(Step 2) Second mutation induced by gamma irradiation

The strains selected in step 1 were cultured in a YM liquid medium for 5 days with shaking. The culture broth was centrifuged using a sterilized centrifuge tube, washed with 0.1 M phosphate buffer solution (pH 7.0) The buffer solution was suspended at an absorbance value of 2.3 at 600 nm and used for mutagenesis by gamma irradiation.

Mutagenesis by gamma irradiation was carried out in the same manner as in step 1.

All of the colonies that were produced were excellent in pigmentation, glossy, stable, and dark pink colonies. The selection procedure in the YD solid medium was repeated three times to obtain pure colonies.

The selected colonies were cultured in each YM liquid medium for 5 days, and then the dried cell mass and the total carotenoid were measured in the same manner as in Reference Examples 2 and 3 to select one strain having the highest pigment yield.

One selected strain was cultivated in a fed-batch manner in the same manner as in Reference Example 1, and the dried cell mass and the total carotenoids were measured in the same manner as in Reference Examples 2 and 3. As a result, the selected strain had a cell yield Yx / s) and the total carotenoid production yield (Yp / x) including astaxanthin pigment of 5,487 占 퐂 / g was found to be 329,220 占 퐂 /? as a whole.

(Step 3) Third mutagenesis by NTG treatment

The strains selected in step 2 were cultured in a YM liquid medium for 5 days with shaking. The culture broth was centrifuged using a sterilized centrifuge tube, washed with 0.1M citrate buffer solution (pH 5.5) And the absorbance value at 600 nm was adjusted to 2.3 so as to be used for mutation by NTG.

250 μl of 1.2 mg / ml NTG was added to 4.8 ml of this suspension and allowed to react for 25 minutes so that 95% or more of the papyrus rhodoma yeast was killed. After the reaction was completed, the reaction was stopped by adding 5% sodium chloride solution. The reaction was washed twice with 0.1 M phosphate buffer (pH 7.0), and 5 ml of YM liquid medium was added to the precipitated yeast for 12 hours. This culture was plated on a YM solid medium and sufficiently cultured at 21 DEG C for 10 days. At this time, the culture without NTG treatment was diluted and cultured in YM agar medium in the same manner to compare the fungicidal rates of the NTG treated group and the non-treated group.

All of the dark pink colonies which were excellent in pigment production, luster and stable than the colony parent strain were selected. The selection procedure in the YD solid medium was repeated three times to obtain pure colonies.

The selected colonies were cultured in each YM liquid medium for 5 days, and then the dried cell mass and the total carotenoid were measured in the same manner as in Reference Examples 2 and 3 to select one strain having the highest pigment yield.

One selected strain was cultivated in the same manner as in Reference Example 1 and cultured in a fed-batch manner. The dried cell mass and the total carotenoid were measured in the same manner as in Reference Examples 2 and 3, and the selected strain had a cell yield Yx / s) of 61 g / ℓ and total carotenoid production yield (Yp / x) including astaxanthin pigment of 5,936 ㎍ / g, indicating a total pigment production of 362,096 ㎍ / ℓ.

(Step 4) inducing a fourth mutation by the silymar treatment

The colonies selected in step 3 were placed in a YM liquid medium, shake-cultured for 5 days at a stirring speed of 150 rpm, and the culture was diluted with a selective medium containing 0.4 mM of a mime-containing YM solid medium showing a killing effect of 85% And cultured at 21 캜 for 10 days until the colonies grew. At this time, the same culture broth was diluted in YM solid medium containing no cymol and cultured in the same manner to compare the fungicidal rates of the siMo treated group and the non-treated group.

Among the colonies produced, all of the dark pink colonies which were excellent in pigment production, shiny and stable than the parent strain were selected. The selection procedure in the YD solid medium was repeated three times to obtain pure colonies.

The selected colonies were cultured in each YM liquid medium for 5 days, and then the dried cell mass and the total carotenoid were measured in the same manner as in Reference Examples 2 and 3 to select one strain having the highest pigment yield.

One selected strain was cultivated in the same manner as in Reference Example 1 and cultured in a fed-batch manner. The dried cell mass and the total carotenoid were measured in the same manner as in Reference Examples 2 and 3, and the selected strain had a cell yield Yx / s) 63 g / ℓ and the total carotenoid production yield (Yp / x) including astaxanthin pigment was 6,573 ㎍ / g, indicating that the total pigment yield was 414,099 ㎍ / ℓ.

One selected strain was designated as Papiarodojima KT-9 and deposited with the Korean Society for Microorganism Preservation, KCCM 10112, on October 7, 1997.

Example 2: Selection of Papiarodojima strain KT-12

(Step 1) 5th mutagenesis by NTG treatment

The Papiarodojima strain KT-9 selected in step 4 of Example 1 was cultured in a YM liquid medium for 5 days with shaking at a stirring speed of 150 rpm. The culture broth was centrifuged using a sterilized centrifuge tube, and 0.1 M citrate buffer solution (pH 5.5), and the suspension was suspended in the same buffer at an absorbance value of 2.3 at 600 nm and used for mutagenesis by NTG treatment.

Mutagenesis by NTG was carried out in the same manner as in step 3 of Example 1.

Among the colonies produced, all of the dark pink colonies which were excellent in pigment production, shiny and stable than the parent strain were selected. The selection procedure in the YD solid medium was repeated three times to obtain pure colonies.

The selected colonies were cultured in each YM liquid medium for 5 days, and then the dried cell mass and the total carotenoid were measured in the same manner as in Reference Examples 2 and 3 to select one strain having the highest pigment yield.

One selected strain was cultivated in the same manner as in Reference Example 1 and cultured in a fed-batch manner. The dried cell mass and the total carotenoid were measured in the same manner as in Reference Examples 2 and 3, and the selected strain had a cell yield Yx / s) of 66 g / ℓ and total carotenoid production yield (Yp / x) including astaxanthin pigment of 6,921 ㎍ / g, indicating a total pigment productivity of 456,786 ㎍ / ℓ.

(Step 2) 6th mutagenesis by beta-ionone treatment

The strain selected in step 2 was placed in a YM liquid medium, shake cultured for 5 days, and then the culture was diluted with a 3.0MM beta-ionone-containing YM solid medium, which had a killing effect of 75% or more, Incubate at 21 ° C for about 10 days until the colonies grow. At this time, the same culture broth was diluted in YM solid medium containing no beta-ionone and cultured in the same manner to compare the fungicidal rates of the beta-ionone treated group and the non-treated group.

Among the colonies produced, all of the dark pink colonies which were excellent in pigment production, shiny and stable than the parent strain were selected. The selection procedure in the YD solid medium was repeated three times to obtain pure colonies.

The selected colonies were cultured in each YM liquid medium for 5 days, and then the dried cell mass and the total carotenoid were measured in the same manner as in Reference Examples 2 and 3 to select one strain having the highest pigment yield.

One selected strain was cultivated in the same manner as in Reference Example 1 and cultured in a fed-batch manner. The dried cell mass and the total carotenoid were measured in the same manner as in Reference Examples 2 and 3, and the selected strain had a cell yield Yx / s) 67 g / ℓ and total carotenoid production yield (Yp / x) including astaxanthin pigment 7,450 ㎍ / g, indicating that the total pigment yield was 499,150 ㎍ / ℓ.

(Step 3) 7th mutation induced by NTG treatment

The strains selected in step 2 were cultured in a YM liquid medium for 5 days with shaking at a stirring speed of 150 rpm. The culture broth was centrifuged using a sterilized centrifuge tube, and a 0.1M citrate buffer solution (pH 5.5) After the cells were washed, they were suspended in the same buffer to an absorbance value of 2.3 at 600 nm and used for mutagenesis by NTG treatment.

Mutagenesis by NTG was carried out in the same manner as in step 3 of Example 1.

Among the colonies produced, all of the dark pink colonies which were excellent in pigment production, shiny and stable than the parent strain were selected. The selection procedure in the YD solid medium was repeated three times to obtain pure colonies.

The selected colonies were cultured in each YM liquid medium for 5 days, and then the dried cell mass and the total carotenoid were measured in the same manner as in Reference Examples 2 and 3 to select one strain having the highest pigment yield.

One selected strain was cultivated in the same manner as in Reference Example 1 and cultured in a fed-batch manner. The dried cell mass and the total carotenoid were measured in the same manner as in Reference Examples 2 and 3, and the selected strain had a cell yield Yx / s) and the total carotenoid production yield (Yp / x) including astaxanthin pigment was 7,817 ㎍ / g, indicating that the total pigment yield was 531,556 ㎍ / ℓ.

(Step 4) Eight-order mutation by treatment with triethylamine

The strain selected in step 3 was placed in a YM liquid medium and shake cultured for 5 days at a stirring speed of 150 rpm. The culture was diluted with a YM solid medium containing 15 mM triethylamine having a killing effect of 85% or more Then, cultivate at 21 캜 for 10 days until the colonies grow. At this time, the same culture medium was diluted in YM solid medium containing no triethylamine and cultured in the same manner, and the fungicidal rates of the triethylamine treatment group and the non-treatment group were compared.

Among the colonies produced, all of the dark pink colonies which were excellent in pigment production, shiny and stable than the parent strain were selected. The selection procedure in the YD solid medium was repeated three times to obtain pure colonies.

The selected colonies were cultured in each YM liquid medium for 5 days, and one strain was selected by measuring the dry cell weight and the total carotenoids in the same manner as in Reference Examples 2 and 3.

One selected strain was cultivated in the same manner as in Reference Example 1 and cultured in a fed-batch manner. The dried cell mass and the total carotenoid were measured in the same manner as in Reference Examples 2 and 3, and the selected strain had a cell yield Yx / s) and the total carotenoid production yield (Yp / x) including astaxanthin pigment of 8,022 占 퐂 / g, which was found to be 561,540 占 퐂 / liter.

(Step 5) The 9th mutation by the 4th NTG treatment

The strains selected in step 4 were cultured in a YM liquid medium for 5 days with shaking at a stirring speed of 150 rpm. The culture broth was centrifuged using a sterilized centrifuge tube and cultured in 0.1 M citrate buffer solution (pH 5.5) After the cells were washed, they were suspended in the same buffer to an absorbance value of 2.3 at 600 nm and used for mutagenesis by NTG treatment.

Mutagenesis by NTG was carried out in the same manner as in step 3 of Example 1.

Among the colonies produced, all of the dark pink colonies which were excellent in pigment production, shiny and stable than the parent strain were selected. The selection procedure in the YD solid medium was repeated three times to obtain pure colonies.

The selected colonies were cultured in each YM liquid medium for 5 days, and one strain was selected by measuring the dry cell weight and the total carotenoids in the same manner as in Reference Examples 2 and 3.

One selected strain was cultivated in the same manner as in Reference Example 1 and cultured in a fed-batch manner. The dried cell mass and the total carotenoid were measured in the same manner as in Reference Examples 2 and 3, and the selected strain had a cell yield Yx / s) 72 g / ℓ and total carotenoid production yield (Yp / x) including astaxanthin pigment was 8,210 ㎍ / g, indicating that the total pigment yield was 591,120 ㎍ / ℓ.

(Step 6) Tertiary mutation by beta-carotene treatment

The strain selected in step 5 was placed in a YM liquid medium, shake cultured at a stirring speed of 150 rpm for 5 days, and then the culture was diluted with 0.05% beta-carotene-containing YM solid medium having a killing efficiency of 95% Lt; RTI ID = 0.0 > 21 C < / RTI > At this time, the same culture broth was diluted in YM solid medium containing no beta-carotene and cultured in the same manner to compare the fungicidal rates of beta-carotene treated group and non-carotene treated group.

Among the colonies produced, all of the dark pink colonies which were excellent in pigment production, shiny and stable than the parent strain were selected. The selection procedure in the YD solid medium was repeated three times to obtain pure colonies.

The selected colonies were cultured in each YM liquid medium for 5 days, and one strain was selected by measuring the dry cell weight and the total carotenoids in the same manner as in Reference Examples 2 and 3.

One selected strain was cultivated in the same manner as in Reference Example 1 and cultured in a fed-batch manner. The dried cell mass and the total carotenoid were measured in the same manner as in Reference Examples 2 and 3, and the selected strain had a cell yield Yx / s) 73 g / ℓ and total carotenoid production yield (Yp / x) including astaxanthin pigment 8,434 ㎍ / g, indicating that the total pigment yield was 615,682 ㎍ / ℓ.

One selected colony was designated as Papiarodojima KT-12 and deposited on October 7, 1997 as the deposit number KCCM-10113 to the Korean Society for Microorganism Conservation.

The pigment productivity of the strains selected in the mutagenesis process is summarized in Table 1 below.

Strain name Cell yield (g / l) Total carotenoid yield (占 퐂 / g yeast dry weight) Pigment Productivity (㎍ / ℓ) ATCC 66272 4 975 3,900 HP-30 (NRRL Y-21359) 60 4,200 252,000 The first mutated strain 58 4,925 285,650 A second mutant strain 60 5,487 329,220 The third mutant strain 61 5,936 362,096 The fourth mutant strain (KT-9) 63 6,573 414,099 5th mutant strain 66 6,921 456,786 6th mutant strain 67 7,450 499,150 7th mutant strain 68 7,817 531,556 8th mutant strain 70 8,022 561,540 9th mutant strain 72 8,210 591,120 The 10th mutant strain (KT-12) 73 8,434 615,682

The papi rhodozyma mutant strains KT-9 and KT-12 of the present invention have a high dry cell mass yield (Yx / s) and a total carotenoid production yield (Yp / x), resulting in a total of about 410,000 / / / l. < / RTI > Which was about 1.6 times and 2.5 times higher than that of the parent strain, Papi Rhodozima HP-30, of 252,000 ㎍ / ℓ.

Claims (4)

Phaphia rhodozyma mutant strain KT-9 (KCCM-10112) with high productivity of astaxanthin. N-methyl-N-nitro-N-nitrosoguanidine (NTG) treatment and thymol treatment are applied to the Papi rhodozyma strain at least once or more in arbitrary order so as to have an extinction rate of 60 to 99.9% The mutant strain of claim 1, Astaxanthin-producing high-papillotyroidism mutant strain KT-12 (KCCM-10113). N-methyl-N-nitro-N-nitrosoguanidine (NTG) treatment, beta-ionone treatment, triethylamine (TEA) treatment, beta-carotene (β-carotene) treatment is carried out at least once each in an arbitrary order so as to have an extinction rate of 60 to 99.9%, respectively.