KR100750810B1 - Streptomyces hygroscopicus atcc 14891 mutant and production method of tacrolimus with it - Google Patents

Abstract

A Streptomyces hygroscopicus ATCC 14891 mutant strain and a production method of tacrolimus by using the same mutant strain are provided to improve productivity and yield of tacrolimus used as an antimicrobial agent and an immunosuppressant. The Streptomyces hygroscopicus subsp. ascomyceticus ATCC 14891 mutant strain(KCTC 10964BP) produces high yield of tacrolimus and is produced by inducing mutation of Streptomyces hygroscopicus ascomyceticus ATCC 14891 with UV(ultraviolet) and NTG(N-methyl-N'-nitro-N-nitrosoguanidine). The tacrolimus is produced by culturing Streptomyces hygroscopicus subsp. ascomyceticus ATCC 14891 mutant strain(KCTC 10964BP) in a medium containing 40-60g/L of dextrin, 15-25 g/L of starch, 1.5-2.5 g/L of yeast extract, 5-50g/L of cottonseed powder, 5-20 ml/L of corn steep liquid(CSL), 0.2g/L of dipotassium hydrogen phosphate, 0.05-0.15mg/L of iron sulfate and 0.5-0.7mg/L of copper sulfate.

Description

Streptomyces hygroscopicus ATCC 14891 mutant and production method of tacrolimus with it}

       1 is a view showing the growth curve of the mutant strain according to the present invention in a 300L fermentation tank,

Figure 2 is a diagram showing the HPLC results of the culture sample produced in 300L fermenter of mutant strain according to the present invention,

3 is a diagram showing the 1H nuclear magnetic resonance spectrum of tacrolimus purified from the mutant strain according to the present invention,

4 is a diagram showing a 13C nuclear magnetic resonance spectrum of tacrolimus purified from the mutant strain according to the present invention,

5 is a diagram showing a COSY nuclear magnetic resonance spectrum of tacrolimus purified from the mutant strain according to the present invention,

6 is a diagram showing an infrared absorption spectrum of tacrolimus purified from the mutant strain according to the present invention,

7 is a diagram showing a mass spectrum of tacrolimus purified from the mutant strain according to the present invention.

       It is an object of the present invention to provide a Streptomyces hygroscopius subspecies Ascomycetigus ATCC 14891 mutant strain having an accession number of KCTC 10964BP and a method of culturing the same and mass-producing tacrolimus from the culture.

Tacrolimus is the Streptomyces genus Streptomyces (Streptomyces It is a macrolide lactone-based antibiotic with a tricyclic ring structure biosynthesized by sp.) and was first discovered in fermentation products of Streptomyces Tsukubaensis screened in Tsukuba, Japan. The substance exhibits pharmacological activity, such as antimicrobial activity and immunosuppressive activity. Specifically, GVHD (graft-versus-host disease) is caused by the prevention and treatment of graft rejection and the donor's immune cells destroyed after bone marrow transplantation, which destroys the tissue of the beneficiary. ), Autoimmune diseases and infectious diseases. It is 100 times more immunosuppressive activity than cyclosporine A, which has been widely used as an immunosuppressant in clinical trials, and has been relatively low in side effects such as renal toxicity, hypertension, and rejection of transplants. Cograf, Ltd.) is increasing the sales of Prograf.

Cyclosporin A, sirolimus, mycopinoleic acid, rapamycin and the like are known to exhibit immunosuppressive effects. Cyclosporin A and taclolimus inhibit transcription factors involved in the transcription of T cell active genes (Emmel, EA, Science ., 246 , pp.1617-1620, 1989), and rapamycin that is structurally similar to tacrolimus is lipoca Inhibition of phosphorus production inhibits T cell activity (Sehgal, SN, J. Antibiot . , 28 , pp. 727-732, 1975).

Streptomyces spp. Is currently known to produce tacrolimus in Streptomyces No.9993 (USP 5,624,842) and Streptomyces genus MA6858 (USP 5,116,756), but the strain has low productivity and is fermented for mass production. It is not desirable for the process.

The present inventors induced artificial mutants using mutagen for mass production of tacrolimus to improve this. Strains include strains which are macrolide in Streptomyces to produce the lactone material MA6858, Streptomyces Libby thiooxidans, streptavidin of ascorbyl MY MY access Hi Gloss subspecies of nose kusu Shetty kusu (Streptomyces hygroscopicus subsp . ascomyceticus ) was used, and mutations were induced by UV irradiation and NTG (N-methyl-N'-nitro-N-nitrosoguanidine) to obtain a mutant strain capable of producing tacrolimus. In order to improve the productivity of tacrolimus using the new strain, it is important to maintain sufficient dissolved oxygen, so that the productivity was examined by adding a constant ratio of starch and dextrin, and the optimum fermentation medium composition for mass production was confirmed.

As such, the strains of Streptomyces hygroscopicus subspecies Ascomycetigus ATCC 14891 with an accession number of KCTC 10964BP capable of mass production of tacrolimus compared to the strains having low tacrolimus productivity were identified, and the culture was carried out. By confirming the method for mass production of tacrolimus from the present invention was completed.

It is an object of the present invention to provide a Streptomyces hygroscopius subspecies Ascomycetigus ATCC 14891 mutant strain having an accession number of KCTC 10964BP and a method of culturing the same and mass-producing tacrolimus from the culture.

In order to achieve the above object, the present invention provides a Streptomyces hygroscopicus subspecies Ascomycetigus ATCC 14891 mutant strain having an accession number of KCTC 10964BP.

The present invention also provides a production method for producing tacrolimus, characterized by culturing Streptomyces hygroscopius subspecies Ascomycetigus ATCC 14891 mutant strain of KCTC 10964BP in a culture medium.

The culture medium is dextrin 40 to 60 g / L, starch 15 to 25 g / L, yeast extract 1.5 to 2.5 g / L, cottonseed flour 5 to 50 g / L, corn steep liquor (CSL) 5 to 20 ml / L and iron sulfide 0.05 To 0.15 mg / L and copper sulfate 0.5 to 0.7 mg / L.

Hereinafter, the present invention will be described in detail.

Variant strains of the present invention can be selected as follows.

Tacrolimus-producing strains were selected by thin layer chromatography (TLC) coated with silver nitrate among the mutant strains obtained by performing primary mutation by UV irradiation with Streptomyces hygroscopius ascomasetiticus ATCC 14891. Tacrolimus productivity was obtained by quantitative analysis using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) among the mutant strains obtained by secondary artificial mutation through (N-methyl-N'-nitro-N-nitrosoguanidine) treatment. This highest mutant strain was selected and named Streptomyces hygroscopius DKT523 (KCTC 10964BP).

The mutant strains had similar physiological, morphological and physiological characteristics to the parent strain Streptomyces hygroscopicus ATCC 14891. In addition, ascomycin production was significantly reduced and tacrolimus production was increased compared to the parent strain. The mutant strains having the characteristics described above were deposited with the Korean Gene Bank according to the Budapest Treaty as 2006.6.30 and were given the accession number of KCTC 10964BP, and their bacteriological properties were as follows.

 characteristic Streptomyces hygroscopius ascomyceticus DKT523 shape mycelium Fall color grey Gram Dyeing positivity Oxygen requirements Aerobic Melanin pigment - Tyrosine breakdown - Explode elements + Starch hydrolysis + Casein hydrolysis + Nitrate reduction + Protease Activity weakness   NaCl 1% +    NaCl 3% +    NaCl 5% - -: Below or negative reaction +: above or positive reaction

Tacrolimus can be produced as described below by culturing Streptomyces hygroscopius subspecies Ascomycetigus ATCC 14891 mutant strain selected as described above with KCTC 10964BP in a fermentation medium.

The carbon source used in the fermentation broth composition for producing the tacrolimus in high yield is not particularly limited, but is preferably water-soluble starch, dextrin, and more preferably starch, species for production in consideration of raw material prices. The medium may be used in combination of starch and dextrin, preferably in a starch: dextrin ratio of 1 to 3: 4 to 6 by weight.

Fermentation medium containing starch and dextrin as carbon source is preferably 15 to 25 g / L starch, 40 to 60 g / L dextrin, 1.5 to 2.5 g / L yeast extract, 5 to 50 g / L cottonseed flour, corn steep liquor (CSL ) 5-20 mL / L, dipotassium hydrogen phosphate 0.2 g / L, iron sulfide 0.05-0.15 mg / L and copper sulfate 0.5-0.7 mg / L.

The present invention provides a method for producing tacrolimus in high yield by culturing the mutant strains. The fermentation culture composition is preferably composed of dipotassium hydrogen phosphate, iron sulfide, copper sulfate, carbon source and nitrogen source. The carbon source is glycerin, maltose, dextrin and starch, preferably dextrin and starch, and the nitrogen source is yeast extract, cottonseed flour, corn steep liquor, soybean hydrolyzate and pea flour, preferably corn steep liquor, cottonseed flour And yeast extracts, more preferably carbon source dextrin and nitrogen source cottonseed flour, most preferably dextrin: cottonseed flour = 5-8: 2-4.

In addition, the state of the spawn when the fermentation is carried out greatly affects the fermentation time and productivity in the main culture, the incubation time in the spawn culture is preferably 24 to 48 hours, the inoculation amount is 3 to 10%.

Hereinafter, a method for producing tacrolimus using the Streptomyces hygroscopius subspecies Ascomycetigus ATCC 14891 mutant having the selected accession number KCTC 10964BP will be described in detail.

The spawn culture of the mutant strains was inoculated in a medium (pH 6.8 ± 0.2) containing 30 g / L of dextrin, 2 g / L of yeast extract, 5-10 g / L of cottonseed flour, 5-10 mL / L of corn steep liquor (CSL), It is preferable to perform 30 to 50 hours, preferably 45 to 50 hours at 25 to 30 ° C at 220 rpm.

The seed solution was made to be 5% and inoculated in a fermenter, and dextrin 50g / L, starch 20g / L, yeast extract 2g / L, cottonseed flour 5-50g / L, corn steep liquor (CSL) 5-20mL / In the medium containing L, iron sulfide 0.1mg / L, copper sulfate 0.6 mg / L while maintaining the initial aeration amount of 1.0 to 1.5vvm, the internal pressure of the fermenter is maintained at 0.3 to 0.2bar, the temperature is 20 to 30 ℃, preferably Fermentation is carried out at 150 to 200rpm or less at 23 to 27 ° C, and the fermentation agitation force is gradually increased stepwise to 200 to 400rpm within 48 to 90 hours of fermentation, and the aeration amount is 0.6 to 1.0vvm while maintaining the internal pressure of the fermentor at 0.3 bar. After fermentation is terminated at 150 to 170 hours of culturing, tacrolimus may be produced, and tacrolimus concentration may be finally quantitatively analyzed to obtain tacrolimus.

The production method of tacrolimus has a high productivity when compared to the Streptomyces Tsukubaensis No.9993 (USP 5,624,842) and Streptomyces genus MA6858 (USP 5,116,756), which is a strain of Tacrolimus genus of the prior art Streptomyces, Based on the optimal culture medium composition of the present invention is characterized in that mass production is possible.

Tacrolimus produced by the method of the present invention can be used as a raw material of the conventional tacrolimus has been used, that is, various pharmaceutical formulations.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited by the following Experimental Examples.

Example  One. Tacrolimus  Selection of production variation

1-1. Variation strain by ultraviolet irradiation DKT  523 separations

Mutant strains with tacrolimus-producing ability were selected by thin layer chromatography (TLC) method by irradiating UV rays of subspecies Ascomyceticus ATCC 14891 of Streptomyces hygroscopius.

First, the subspecies of Streptomyces hygroscopicus subspecies Ascomyceticus ATCC 14891 were incubated in a slope medium for 3 days at 28 ° C. After serial dilution with dilution 10 ^-2 , 10 ^-3 , preincubation was performed at 28 ° C. for 2 hours. Two 20W ultraviolet lamps with a wavelength of 280nm were irradiated for 115 seconds in a sterile box installed at a distance of 65cm, at which time the mortality was 99.9%. Shade, preserve for 2 hours at 4 ° C, purify for 2 hours to prevent repair by immediate photorecovery, 4 days at 28 ° C, 6 days in 250 ml baffle flask made of production medium by collecting viable colonies By culturing, strains with tacrolimus producing ability were selected. The first selected mutants were NTG (N-methyl-N'-nitro-N-nitrosoguanidine) using a second mutation was carried out in the same manner as the UV irradiation method at 70% mortality. The selected strains were subjected to fermentation culture in a baffle flask containing a liquid medium to select mutants that produce tacrolimus through HPLC quantitative analysis to confirm the stability of the strains in several replicates (see Table 2 medium composition).

badge Furtherance Reputation and Slope Badges Glucose 10 g / L, yeast extract 3 g / L, malt extract 3 g / L, peptone 5 g / L, agar 20 g / L Preculture Badge Glucose 10 g / L, yeast extract 3 g / L, malt extract 3 g / L, peptone 5 g / L Production badge Dextrin 70 g / L, corn steep liquor 10 ml / L, cottonseed flour 30 g / L, yeast extract 2 g / L, dipotassium hydrogen phosphate 0.2 g / L, iron sulfide 0.0001g / L, copper sulfate 0.0006g / L

1-2. Mutant strain screening

In Example 1-1, the mutant strain having the ability to generate tacrolimus using the mutagen was analyzed by a silver ion-coated thin layer chromatography (TLC) method. The TLC plate was coated on a 0.3 M silver nitrate (AgNO ₃ ) solution using silica gel 60F ₂₅₄ for 5 minutes and dried at 100 ° C. for 5 hours to test tacrolimus production and productivity. The development of 20 μl sample for TLC analysis was carried out using a developing solvent (Hexane: Ethyl acetate = 2: 1) for 5 minutes and the color development was anisealdehyde (25ml H ₂ SO _{4 ,} 675ml Etyl alcohol, 18.5ml p-anisaldehyde , 7.5 ml glacial acetic acid) was used to select tacrolimus producing strains.

1-3. Mutant DKT  Characterization of 523

The culture, morphological and physiological characteristics of the mutant strain DKT 523 selected in Example 1-2 were investigated. The cell wall of the mutant strain DKT 523 was Type 1 (LL-diaminopimelic acid) observed. Growth temperature was 18-33 ° C., optimum temperature was 28 ° C. Ascomyceticus ATCC 14891, a subspecies of Streptomyces hygroscopius, produces ascomycin, and the mutant DKT 523 produced ascomycin and tacrolimus. Carbon source availability experiments were carried out according to the method of Freedam and High Trip (Pridham, TG and D. Gotrlib., J. Bacteriol. , 56 , pp.107-114, 1948) and observed after 2 weeks incubation at 28 ℃ It is shown in Table 3 and Table 4 below.

 characteristic Streptomyces hygroscopius ascomyceticus ATCC314891 Streptomyces hygroscopius ascomyceticus DKT 523 shape mycelium mycelium Color of fungi grey grey Gram Dyeing positivity positivity Oxygen requirements Aerobic Aerobic Melanin pigment - - Tyrosine breakdown - - Explode elements + + Starch hydrolysis + + Casein hydrolysis + + Nitrate reduction + + protease activity Very weak weakness   NaCl 1% + +    NaCl 3% + +    NaCl 5% + - -: Below or negative reaction +: above or positive reaction

Availability of Mutant Carbon Sources Carbon source Streptomyces hygroscopius ascomyceticus DKT523 glucose + Arabinos - Sucrose - Maltose ++ Lactose - Mannose - dextrin ++ Starch ++ -: utilization negative ±: utilization doubtful +: positive utilization ++: strongly positive utilization

1-4. Mutant DKT  523 Tacrolimus  Production confirmation

As a tacrolimus quantitative method, a column (Optimapak C18 4.6 × 250mm, RStecch co., Ltd.) and HPLC (Waters) were used, and a standard was performed using a Prograf produced by Fujisawa. The fermentation broth of mutant strain DKT 523 was extracted with 50% methanol for 30 minutes and then experimented with acetonitrile and 0.1% phosphoric acid solution in a ratio of 65:35 as a mobile phase. As shown in FIGS. 2 to 7, the OH stretching (OH strech) is 3449 cm ⁻¹ , the CH stretching is 2927 cm ⁻¹ , and the C = O stretching motion is shown in the infrared spectral spectrum (FT-IR). C = O strech) to 1741 ㎝ ^-1, was confirmed ^{1639 ㎝ -1, 1H, 13C,} CH COSY nuclear magnetic resonance to perform U.S. Patent No. 4,894,366 and the literature (DF Mierke et al., Helvetica . chemica. acta ., pp.1027-1047, 1991) was compared to the standard spectrum indicated the molecular weight was confirmed by mass spectrometry and was determined as 803 that tacrolimus.

Example  2. Tacrolimus  Screening of Media Compositions for High Production

2-1. Single in flask culture As a carbon source Tacrolimus  Production comparison

Tacrolimus production mutant DKT 523 was used to compare the production of a single carbon source, glucose, arabinose, sucrose, maltose, lactose, mannose, dextrin, starch and the like. At this time, 2g of yeast extract, 20g of cottonseed flour, 10ml of corn steep liquor (CSL), and 2g of calcium carbonate were added and cultured at 28 ° C. at 220 rpm for 6 to 8 days. Carbon source concentration is shown in Table 5 below the production of DKT 523 of the best results after experimenting at various concentrations, such as 10 to 100 g.

Carbon source Production amount (mg / ℓ) glucose 40 Sucrose 15 Maltose 50 dextrin 130 Starch 90

As shown in Table 5, when 70 g / l of dextrin was used as a single carbon source, tacrolimus production was 130 mg / l, while the fermentation period was 6 days, which was 1 to 2 days shorter than that of other carbon sources. When starch is used, the fermentation period is 8 days and the fermentation broth is highly viscous. Sucrose and glucose were significantly inhibited in growth. Starch was added to delay the rapid rise of pH in the medium, prolong the incubation time by one to two days, and increase tacrolimus production. The concentration of tacrolimus according to the addition ratio of dextrin and starch was investigated while maintaining the carbon source concentration at 7%, and the optimum carbon source addition ratio was found to be dextrin: starch = 5: 2.

2-2. In flask culture Tacrolimus  Effect of Trace Metal Ions on Production

In order to confirm the effects of each of the trace elements, and to find the optimum concentration of each trace element, an experiment using a plaquett-Burman factor design was performed and the results are shown in Table 6 below. Cobalt salt (CoCl ₂ ) showed a strong side effect of trace elements, and CuSO ₄ and FeSO ₄ showed the strongest positive effect. Based on this, CuSO ₄ was added at a final concentration of 0.1 mg / l and FeSO ₄ was added at 0.6 mg / l, and the result was incubated for 7 days. Was increased.

Added trace element Yield (mg / L) CoCl ₂ 85 CuSO ₄ 145 FeSO ₄ 165 CuSO _{4 ,} FeSO ₄ 190

2-3. Carbon source  According to the addition ratio between nitrogen sources Tacrolimus  Check yield

To determine the optimal concentration of nitrogen source on tacrolimus production, 70 g / l of dextrin as a carbon source and 5 to 20 ml / l of corn steep liquor as a carbon source, 0 to 50 g / l of cottonseed flour, and 2 g / y of yeast extract L and iron sulfide and copper sulfate were combined with trace elements and incubated in a 500 ml flask containing 50 ml for 7 days at 26 ° C. at 220 rpm for quantitative analysis. The change according to the ratio of corn steep liquor did not show a big difference, and the results according to the concentration of cottonseed flour showed that the optimum concentration was 30 g / ℓ. It was confirmed. Therefore, it was confirmed that the medium addition ratio of dextrin and cottonseed flour which can maximize the production of tacrolimus is 7 to 3 (see Table 7).

Streptomyces hygroscopius ascomyceticus DKT523 Dextrin: Starch: Cottonseed Powder Addition 5: 2: 0 5: 2: 1 5: 2: 2 5: 2: 3 5: 2: 4 5: 2: 5 Production amount (mg / ℓ) 52 113 181 220 142 48

Example  3. Tacrolimus  Production variation DKT  523 Fermentation Process

The fermentation productivity of tacrolimus was confirmed in mutant strain DKT 523 at 25 ° C. with 160 L of optimized medium identified in Example 2 in a 300 L fermenter (Kobiotech Co., Ltd).

The spawn culture was carried out in two stages. The primary preculture was incubated for 48 hours at 28 ° C. and 220 rpm in 8 500 ml flasks. The primary seed was inoculated in a 30 L fermenter with 16 L of culture to 5%. Fermentation Batch fermentation was carried out in a 300 L fermenter in a 300 L fermenter containing 160 L of the culture medium at a fermentation condition of 150 to 200 rpm, 1.0 to 1.5 vvm, and internal pressure of 0.3 to 0.5 bar at 25 ° C. in an initial fermentation condition. The cell growth reached a maximum of 21 g / L in dry cell weight (DW) at 90 hours, and the packed mycellium volume (PMV) continued to increase thereafter, approaching 90% at 140 hours.

The maximum cell specific growth rate of the production strain was the highest at less than 48 hours at the beginning of culture. And the fermentation time from 24 to 72 hours to increase the oxygen utilization rate (OUR) to the maximum by increasing the stirring speed of the fermenter to 200 to 400rpm, maintain the internal pressure of the fermenter at 0.3bar and the aeration amount is 0.6 to 1.0vvm to control the excess oxygen partial pressure Adjusted.

In the carbon source utilization of the strain, dextrin 50 g / L and starch 20 g / L were consumed from the low molecular dextrin at the beginning of the culture, and were depleted to less than 20 g / L after 120 hours of culture. At the end of the incubation period, total sugars were less than 10 g / L. In batch fermentation, the pH of the fermentation broth began to increase to nearly 5.2 in 24 hours. As shown in Figure 1, the production of tacrolimus increased as the pH was increased, the production of tacrolimus no longer increased above pH 7.6.

When the amount of oxygen partial pressure (dissolved oxygen) was measured in batch fermentation according to the time period, dissolved oxygen was maintained at almost 0 state at 19 hours of cultivation, and gradually increased to its peak, and remained constant at almost 80% DO after 96 hours of cultivation. . Indeed, if the level of D.O (%) was kept constant at about 24 hours of cultivation, the amount of dissolved oxygen was not short enough, and the production of tacrolimus was a typical secondary metabolite gradually increasing after 72 hours of culture. Tacrolimus production increased dramatically after the end of cell proliferation, when carbon and other nutrients fell to certain levels. In addition, there was a gradual increase in the number of water-soluble pigments, the same secondary metabolite. After fermentation was terminated at 164 hours of cultivation, the tacrolimus concentration was quantitatively analyzed to obtain a final production yield of 420 mg / L.

As described above, the present invention is streptomyces hygroscopicus subspecies Ascomycetigus ATCC 14891 mutant having a deposit number KCTC 10964BP capable of mass production compared to existing tacrolimus producing strains, and cultured it and tacrolimus (tacrolimus) from the culture. Provides a method of mass production.

Claims (4)

      Streptomyces hygroscopius subspecies Ascomycetigus ATCC 14891 mutant strain KCTC 10964BP that produces high yield of tacrolimus. A method of producing tacrolimus, characterized in that the culturing strain of claim 1 in a culture. According to claim 2, wherein the culture composition of the dextrin 40 to 60 g / L, starch 15 to 25 g / L, yeast extract 1.5 to 2.5 g / L, cottonseed flour 5 to 50 g / L, corn steep liquor (CSL) 5 To 20 ml / L, dipotassium hydrogen phosphate 0.2 g / L and 0.05 to 0.15 mg / L iron sulfide and 0.5 to 0.7 mg / L copper sulfate. The method of claim 2, wherein the culture is maintained in the fermenter with an initial aeration amount of 1.0 to 1.5vvm while maintaining the internal pressure of the fermenter at 0.3 to 0.2bar, the temperature is 23 to 27 ℃ fermentation at 150 to 200rpm or less, And gradually increasing the fermentation agitation force stepwise to 200 to 400 rpm within 48 to 90 hours of the fermentation, maintaining the aeration amount at 0.6 to 1.0 vvm while maintaining the internal pressure of the fermenter at 0.3 bar.

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