WO2005063963A1 - Microorganism producing tacrolimus and mass-productive method tacrolimus using the same - Google Patents

Microorganism producing tacrolimus and mass-productive method tacrolimus using the same Download PDF

Info

Publication number
WO2005063963A1
WO2005063963A1 PCT/KR2004/003518 KR2004003518W WO2005063963A1 WO 2005063963 A1 WO2005063963 A1 WO 2005063963A1 KR 2004003518 W KR2004003518 W KR 2004003518W WO 2005063963 A1 WO2005063963 A1 WO 2005063963A1
Authority
WO
WIPO (PCT)
Prior art keywords
tacrolimus
strain
mass
present
streptomyces
Prior art date
Application number
PCT/KR2004/003518
Other languages
French (fr)
Inventor
Tae-Won Kang
Byoung-Taek Choi
Hyung-Soo Kim
Seong-Seon Yu
Original Assignee
Ckd Bio Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ckd Bio Corp filed Critical Ckd Bio Corp
Publication of WO2005063963A1 publication Critical patent/WO2005063963A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces

Definitions

  • the present invention relates to a strain producing tacrolimus, more precisely, a Streptomyces clavuligerus strain mass-producing tacrolimus, an immunosuppressant .
  • An immunosuppressant is clinically used to inhibit unnecessary immune reaction. For example, it is used for the post-care of organ transplantation, for the prevention of Rh hemolytic disease of the newborn, and for the treatment of autoimmune disorder. Precisely, when an organ or a part of organ is transplanted, immune reaction to refuse the donor's organ or tissue is shown inside of a recipient, which has to be inhibited for the success of the transplantation. Thus, an immunosuppressant is inevitable for organ allotransplantation. Cyclosporin is the most representative immunosuppressant today.
  • Cyclosporin is a kind of natural lipophilic cyclic oligopeptides (11-mers) produced from a fungus Tolypocladium infla tum Gams ' ' or other fungi imperfecti.
  • the major component of cyclosporin is cyclosporin A, which is a well-known immunosuppressant specifically inhibiting adaptive immune response by blocking the T cell activation (Kahan, B.D., New Engl . J. Med., 321: 1725-1738, 1989).
  • cyclosporin A which is a well-known immunosuppressant specifically inhibiting adaptive immune response by blocking the T cell activation (Kahan, B.D., New Engl . J. Med., 321: 1725-1738, 1989).
  • it has problems of inducing nephrotoxicity, hypertension and posttransplantation lymphoproliferative disorders.
  • the second most popular immunosuppressants are tacrolimus, sirolimus, mycophenolate, etc.
  • tacrolimus has been attracting our attention most because it shows less transplantation rejection, lower frequency of hypertension, in particular, less side effects with the equal amount of other immunosuppressants .
  • Tacrolimus was first found in fermentation broth of Streptomyces tsukubaensls, a microorganism isolated from soil sample taken from northern Japan, Tsukubae, and it was confirmed to be an antibiotics belonging to macrolides showing immune response inhibiting effect.
  • Tacrolimus as an immunosuppressant, has been used for the post-treatment of kidney or liver transplantation in order to prevent undesirable immune rejection. It was approved by FDA in 1993 and began to be on market in 1994 by Fujisawa Healthcare Co. in the brand name of Prograf in the pharmaceutical forms for oral administration and for inj ection.
  • the informed microorganisms producing tacrolimus are
  • Streptomyces tsukubaensis No.9993 US Patent No. 5,624,842
  • Streptomyces sp. MA 6858 US Patent No. 5,116,756.
  • yield by those microorganisms is very low, indicating that it is not economical to use those microorganisms for the production of tacrolimus.
  • the present inventors have searched and identified a novel microorganism producing tacrolimus that is suitable for the mass-production.
  • the present inventors further have completed this invention by confirming that tacrolimus can be mass-produced and purified from the microorganism.
  • the present invention provides a Streptomyces clavuligerus strain mass-producing tacrolimus and a method for mass-production of tacrolimus by using the same.
  • the present invention provides a Streptomyces clavuligerus strain producing tacrolimus represented by formula 1.
  • the present invention also provides a method for mass-production of tacrolimus including the culture of the above strain in a culture medium.
  • the present invention provides a Streptomyces clavuligerus strain producing tacrolimus represented by formula 1.
  • the strain is preferably a Streptomyces clavuligerus CKD 1119 (Accession No: KCTC 10561BP) .
  • a novel strain of the present invention was obtained from screening of a microorganism producing tacrolimus from sample soil, for which bioassay and immunosuppressive activity test were performed based on agar diffusion method using Aspergillus niger ATCC 6275, whose growth is characteristically inhibited by an immunosuppressant, as a test strain.
  • the physiological characteristics of the strain were also investigated by doing research on growth temperature, melanin pigment, starch hydrolysis, reduction of nitrogen group, gelatin liquefaction, coagulation of litmus milk and peptonization, formation of hydrogen sulfide and clavulanic acid formation, resulting in the confirmation that the strain has the same physiological characteristics as Streptomyces clavuligerus has (see Table 3) .
  • the utilization of carbon source was also investigated, resulting in the similar aspects to that shown by Streptomyces clavuligerus, except the use of mannose and glucose (see Table 4).
  • genomic DNA of the strain of the present invention was separated and a whole base sequence of 16S rDNA was investigated.
  • the strain of the present invention was confirmed to have 98.39% homology with Streptomyces clavuligerus NRRL3585, a standard strain of Streptomyces clavuligerus . But, there was a little difference between the two strains in the color of medium made by a soluble pigment secreted from a strain and in the production of tacrolimus.
  • the present inventors named the strain of the invention x Streptomyces clavuligerus CKD 1119' and then deposited it at Korean Collection for Type Cultures (KCTC) of Korea Research Institute of Bioscience and Biotechnology (KRIBB) on December 5, 2003 (Accession No: KCTC 10561BP) .
  • the present inventors compared Streptomyces clavuligerus CKD 1119 of the invention with Streptomyces tsukubaensis No.9993 and Streptomyces sp. MA 6858, which are well-known strains to produce tacrolimus, in physiological and chemical characteristics. As a result, those three strains showed same cell wall composition that is type I and had LL type diaminopimelic acid equally, but they showed differences in cultural, morphological and physiological characteristics, in addition to the differences in the utilization of a carbon source and fermentation product. The above results indicate that the strain of the present invention belongs to the same genus where the common strains producing tacrolimus belong, but it belongs a different species from those strains.
  • a substance having immunosuppressing activity which is produced in the strain of the invention, was separated, and then identified by nuclear magnetic resonance spectrum, infrared spectrum and high performance liquid chromatography (HPLC) .
  • the substance having immunosuppressing activity was confirmed to be tacrolimus represented by formula 1 (see Fig. 1 ⁇ Fig. 4) .
  • the present invention also provides a method for mass-production of tacrolimus by the culture of
  • the medium for the culture is preferably supplemented with malonic acid, ethanol, methionine, carbon source and nitrogen source.
  • a carbon source one or more substances are preferably selected from a group consisting of starch, glucose, corn oil, glycerol, maltose, mannose and inositol, and starch, glucose and corn oil are more preferred.
  • a nitrogen source one or more substances are selected preferably from a group consisting of cottonseed flour, corn steep liquor, corn steep powder, soybean flour, peptone and yeast extract, and among them, cotton seed flour and corn immersion solution are more preferred.
  • seed culture solution containing Streptomyces clavuligerus CKD 1119 was inoculated to a fermentor containing the above-mentioned medium (see Table 5) . Then, the culture solution was filtered and extraction was performed to obtain oily residue. A fraction including a target compound was purified by column chromatography, resulting in pure tacrolimus.
  • Fig. 1 is a 13 C nuclear magnetic resonance spectrum of tacrolimus produced in the strain of the present invention.
  • Fig. 2 is a 1 H nuclear magnetic resonance spectrum of tacrolimus produced in the strain of the present invention.
  • Fig. 3 is an infrared spectrum of tacrolimus produced in the strain of the present invention.
  • Fig. 4 is a high performance liquid chromatography
  • Example 1 Isolation and selection of a microorganism Agar plate dilution method was used to separate a microorganism of the present invention. Particularly, 1 kg of sample soil taken from Boryeong city, Chungnam, Korea was put in a sterilized test tube and sterilized water was added thereto. After 10 minutes of settling, supernatant was obtained.
  • the obtained supernatant was 100-fold diluted with sterilized water, and then 0.1 mi of the diluted solution was distributed onto yeast-malt extract agar (yeast extract 4 q/l , malt extract 10 q/i , glucose 4 q/l , agar 20 q/l , distilled water 1 I , pH 6.8 ⁇ 7.0) in petri dish.
  • yeast-malt extract agar yeast extract 4 q/l , malt extract 10 q/i , glucose 4 q/l , agar 20 q/l , distilled water 1 I , pH 6.8 ⁇ 7.0
  • the solution was cultured at 28 °C for 21 days, after which a colony formed on the yeast-malt extract agar was picked up with a sterilized needle.
  • the obtained colony was transferred to a fresh yeast-malt extract agar at regular interval, followed by sub-culture. The colonies proliferated during the sub-culture were further cultured at 28 ° C for 10
  • the spore suspension (1 x 10 7 ⁇ 10 8 cells/m ⁇ ) of Aspergillus niger ATCC 6275 was distributed by 0.5 mi .
  • Aspergillus niger ATCC 6275 was used as a test fungus of the present invention because its' growth is inhibited by an immunosuppressant.
  • colonies were additionally cultured for 2 more days at 28°C. Then, those colonies (276) especially inhibiting greatly the growth of Aspergilus niger ATCC 6275 were selected.
  • T-cells of mouse spleen were distributed into a 96-well plate for microculture containing RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with heat-inactivated 10% fetal calf serum by 200 ⁇ i per each well with the density of 0.5 x 10 6 cells/mi . Extraction was performed by using methanol from the culture solutions of primarily selected 127 strains, followed by concentration of the extract.
  • the concentrated extract was distributed into wells, which were additionally supplemented with 250 nq/ i of ionomycin (a kind of ionophore, a conductor of Ca 2+ ) and 10 nq/mi of PMA (Peptonized Milk Agar; peptonized milk 1 g, distilled water 1 I ) liquid medium. Reaction with radioisotope 3 H- TdR (Thimidine deoxyribose) (S .A. (Specific activity) 5 Ci/mmol; Cone. 1 uCi/m#) was induced for about 5 hours, followed by further culture at 37 ° C for 48 hours.
  • the culture solution was filtered by glass wool to collect cells, and then radioassay was performed using a spectrophotometer . At that time, radioactivity of a control group that excluded a sample of the culture solution was regarded as 100%, providing a standard for the comparison with radioactivity of other experimental groups. For the confidence of the test results, all the above experiments were repeated three times.
  • CKD 1119 isolated in the above example 2 were also investigated. Particularly, isolated strains were inoculated onto yeast-malt extract agar, potato dextrose agar, oatmeal agar, Czapek agar, glucose-asparagine agar, peptone-dextros agar and skim milk agar, followed by culturing at 28 ° C for 7 - 14 days. Then, growth, spore formation, colony color and soluble pigment were investigated (Table 1)
  • yeast-malt extract agar medium in which the colony color was yellow and a secreted soluble pigment was dark orange.
  • CKD 1119 was cultured in yeast-malt extract agar at 28 °C for 14 days. Then, observation was carried out under optical and electron microscope. As a result, it was confirmed that a rectiflexible spore chain was formed and the surface of the spore was smooth.
  • NRRL 3585 ATCC 27064
  • a standard strain of Streptomyces clavuligerus and a novel strain of the present invention were respectively inoculated onto yeast-malt extract agar, followed by culturing at 28 ° C for 14 days. During the culture, the sizes, shapes and colors of colonies and the color change of the medium were investigated (Table 2) .
  • Physiological characteristics of a novel strain of the present invention were compared with that of Streptomyces clavuligerus NRRL 3585.
  • the strains were cultured in a medium composed of 1 ( of distilled water, 15 g of glycerol, 38.5 g of soybean flour, 23 g of rice bran oil, 2 g of KH 2 P0 4 and 21 g of MOPS (3- [N-Morpholino] propanesulfonic acid sodium salt, CH ⁇ 4 N0SNa) , at 25 ° C for 6 days.
  • genomic DNA of the strain was separated and a whole nucleotide sequence of 16S rDNA was analyzed.
  • genomic DNA was separated by using the method of Rochell, et al (Rochell, P.A., et al . , FEMS Microbiol . Lett . , 100: 59-66, 1992), then PCR (polymerase chain reaction; 94 ° C 1 minute, 52-56 ° C 1 minute, 72 °C 2 minutes) was performed to synthesize ribosomal subunit gene (16S rDNA) .
  • Nucleotide sequence of the synthesized DNA was determined by using Tag Dye Deoxy Terminator Cycle Sequencing Kit.
  • the present inventors named the strain of the invention ⁇ Streptomyces clavuligerus CKD 1119' and then deposited it at Korean Collection for Type Cultures (KCTC) of Korea Research Institute of Bioscience and Biotechnology (KRIBB) on December 5, 2003 (Accession No: KCTC 10561BP) .
  • KCTC Korean Collection for Type Cultures
  • KRIBB Research Institute of Bioscience and Biotechnology
  • Streptomyces tsukubaensis No.9993 and Streptomyces sp. MA 6858 are well-known tacrolimus producing strains.
  • Patent No. 5,624,842 characteristics of Streptomyces tsukubaensis No.9993
  • US Patent No. 5,116,756 characteristics of Streptomyces sp. MA 6858.
  • Cell wall compositions of those three strains were all judged as type I, and LL type diaminopimelic acid was equally observed. However, other characteristics were different among them. Precisely, cultural, morphological and physiological characteristics and the carbon source utilization as well as fermentation products were all investigated and compared among them.
  • Streptomyces tsukubaensis No.9993 was negative to the reaction but Streptomyces clavuligerus CKD1119 strain of the present invention was positive to the reaction. While Streptomyces tsukubaensis No.9993 strain showed positive response to milk peptonization, Streptomyces clavuligerus CKD1119 of the present invention was negative. Formation of hydrogen sulfide was also investigated. Streptomyces sp. MA6858 strain was positive to the formation of hydrogen sulfide, but Streptomyces clavuligerus CKD1119 of the present invention was negative to the reaction.
  • Streptomyces tsukubaensis No.9993 strain was negative to use inositol and had a small chance of using sucrose
  • Streptomyces clavuligerus CKD1119 of the present invention had a small chance of using inositol and was negative to use sucrose.
  • Streptomyces sp. MA 6858 strain was positive to use lactose and glucose
  • Streptomyces clavuligerus CKD1119 of the present invention was negative to use lactose and had a just small chance of using glucose.
  • Streptomyces tsukubaensis No.9993 and Streptomyces sp. MA6858 produced tacrolimus only
  • Streptomyces clavuligerus CKD1119 of the present invention produced not only tacrolimus but also clavulanic acid.
  • Streptomyces clavuligerus CKD1119 strain of the present invention is a novel strain that is different from both of the two tacrolimus producing common strains, Streptomyces tsukubaensis No.9993 and Streptomyces sp. MA6858.
  • the present inventors identified an immunosuppressant produced by CKD 1119 strain secondly selected in the above example 2.
  • the strain was cultured as a monocolony in a petri dish containing 30 mi of yeast-malt extract agar (starch 10 q/l , yeast extract 4 q/l , malt extract 10 q/l , agar 20 q/l , distilled water 1 , pH 6.8 - 7.0), at 28 ° C for 7 - 14 days.
  • the monocolony was suspended in 1.5 mi of saline, which was distributed onto several petri dishes containing the solid medium, followed by further culture at 28 °C for 7 - 14 days.
  • the immunosuppressant produced from the strain of the present invention was tacrolimus.
  • Major function groups of tacrolimus such as hydroxyl group (3447 cm -1 ) , carbonyl group (1743 cm -1 ) and carbon double bond (1650 cm -1 ) were confirmed through infrared spectrum.
  • 13 C and 1 H nuclear magnetic resonance spectrums were compared with the spectrum of standard material (US Patent No. 5,624,842), resulting in the confirmation that the immunosuppressant was tacrolimus (Fig. 1 - Fig. 4).
  • the tacrolimus productivity of the novel strain Streptomyces clavuligerus CKD1119' of the present invention was observed in a jar fermenter. Particularly, 5 I round flask was supplemented with 600 mi of germination culture medium, which was sterilized at 121T for 40 minutes. Then, spore suspension was inoculated thereto (final cone. 1% v/v) . The inoculated strain was shaking-cultured at 28 °C for 48 hours with 120 rpm, resulting in the preparation of germination culture solution. The germination culture solution was inoculated into a 30 I fermenter containing 18 I of germination culture medium (final cone. 3.3% v/v).
  • Tacrolimus produced by Streptomyces clavuligerus CKD1119 of the present invention was separated by the method described in example 6 and then purified by the following method. Particularly, 18 I of the culture solution obtained in example 6 was filtered with 0.3 kg of diatomite, followed by extraction from mycelium cake with 22 I of ethyl acetate. As a result, 22 I of extract was obtained. The ethyl acetate extract obtained from the mycelium was concentrated under reduced pressure to obtain oily phase residue. The residue was mixed with acidic silica gel (special grade 12 silica gel, Fuji Devision Co.) weighing double the residue. The mixture was slurrized in ethyl acetate.
  • acidic silica gel special grade 12 silica gel, Fuji Devision Co.
  • the microorganism of the present invention enables economical mass-production of tacrolimus, comparing to the conventional tacrolimus producing strains. And, highly qualified tacrolimus can be separated from the microorganism of the present invention, so that it can contribute to the production of an immunosuppressant which is essential for the prevention and the treatment of graft rejection accompanied with organ transplantation, graft-versus-host disease caused by bone marrow transplantation, autoimmune diseases, inflammatory diseases, etc.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention relates to a tacrolimus producing strain, more precisely, a Streptomyces clavuligerus strain mass-producing tacrolimus represented by formula 1 and a method for mass-production of tacrolimus by using the same. The strain of the present invention enables mass-production of highly qualified tacrolimus which can be used as an immunosuppressant, so that it can be effectively used for the preparation of a therapeutic agent for organ transplantation, Rh hemolytic disease of the newborn, autoimmune diseases, etc.

Description

.[DESCRIPTION]
[invention Title] MICROORGANISM PRODUCING TACROLIMUS AND MASS- PRODUCTIVE METHOD OF TACROLIMUS USING THE SAME
[Technical Field] The present invention relates to a strain producing tacrolimus, more precisely, a Streptomyces clavuligerus strain mass-producing tacrolimus, an immunosuppressant .
[Background Art] An immunosuppressant is clinically used to inhibit unnecessary immune reaction. For example, it is used for the post-care of organ transplantation, for the prevention of Rh hemolytic disease of the newborn, and for the treatment of autoimmune disorder. Precisely, when an organ or a part of organ is transplanted, immune reaction to refuse the donor's organ or tissue is shown inside of a recipient, which has to be inhibited for the success of the transplantation. Thus, an immunosuppressant is inevitable for organ allotransplantation. Cyclosporin is the most representative immunosuppressant today. Cyclosporin is a kind of natural lipophilic cyclic oligopeptides (11-mers) produced from a fungus Tolypocladium infla tum Gams '' or other fungi imperfecti. The major component of cyclosporin is cyclosporin A, which is a well-known immunosuppressant specifically inhibiting adaptive immune response by blocking the T cell activation (Kahan, B.D., New Engl . J. Med., 321: 1725-1738, 1989). However, it has problems of inducing nephrotoxicity, hypertension and posttransplantation lymphoproliferative disorders. The second most popular immunosuppressants, next to cyclosporin A, are tacrolimus, sirolimus, mycophenolate, etc. Among them, tacrolimus has been attracting our attention most because it shows less transplantation rejection, lower frequency of hypertension, in particular, less side effects with the equal amount of other immunosuppressants .
Tacrolimus was first found in fermentation broth of Streptomyces tsukubaensls, a microorganism isolated from soil sample taken from northern Japan, Tsukubae, and it was confirmed to be an antibiotics belonging to macrolides showing immune response inhibiting effect. Tacrolimus, as an immunosuppressant, has been used for the post-treatment of kidney or liver transplantation in order to prevent undesirable immune rejection. It was approved by FDA in 1993 and began to be on market in 1994 by Fujisawa Healthcare Co. in the brand name of Prograf in the pharmaceutical forms for oral administration and for inj ection. The informed microorganisms producing tacrolimus are
Streptomyces tsukubaensis No.9993 (US Patent No. 5,624,842) and Streptomyces sp. MA 6858 (US Patent No. 5,116,756). However, yield by those microorganisms is very low, indicating that it is not economical to use those microorganisms for the production of tacrolimus.
Thus, in order to overcome such disadvantages, the present inventors have searched and identified a novel microorganism producing tacrolimus that is suitable for the mass-production. The present inventors further have completed this invention by confirming that tacrolimus can be mass-produced and purified from the microorganism.
[Disclosure] [Technical Problem] It is an object of the present invention to provide a Streptomyces clavuligerus strain mass-producing tacrolimus and a method for mass-production of tacrolimus by using the same. [Technical Solution] In order to achieve the above object, the present invention provides a Streptomyces clavuligerus strain producing tacrolimus represented by formula 1. The present invention also provides a method for mass-production of tacrolimus including the culture of the above strain in a culture medium.
[Formula l]
Figure imgf000006_0001
Hereinafter, the present invention is described in detail. The present invention provides a Streptomyces clavuligerus strain producing tacrolimus represented by formula 1. At this time, the strain is preferably a Streptomyces clavuligerus CKD 1119 (Accession No: KCTC 10561BP) . A novel strain of the present invention was obtained from screening of a microorganism producing tacrolimus from sample soil, for which bioassay and immunosuppressive activity test were performed based on agar diffusion method using Aspergillus niger ATCC 6275, whose growth is characteristically inhibited by an immunosuppressant, as a test strain. Cultural, morphological and physiological characteristics of the selected microorganism were investigated along with the chance of using a carbon source. As a result, a novel strain of the present invention was identified to be a Streptomyces clavuligerus . Precisely, from the research on the medium specific growth (see Table 1), cell wall composition of Type I , diaminopimelic acid in the form of LL and morphology of a colony (see Table 2), the strain of the present invention was confirmed to have the same morphological characteristics as Streptomyces sp. has. The physiological characteristics of the strain were also investigated by doing research on growth temperature, melanin pigment, starch hydrolysis, reduction of nitrogen group, gelatin liquefaction, coagulation of litmus milk and peptonization, formation of hydrogen sulfide and clavulanic acid formation, resulting in the confirmation that the strain has the same physiological characteristics as Streptomyces clavuligerus has (see Table 3) . The utilization of carbon source was also investigated, resulting in the similar aspects to that shown by Streptomyces clavuligerus, except the use of mannose and glucose (see Table 4). For more accurate identification, genomic DNA of the strain of the present invention was separated and a whole base sequence of 16S rDNA was investigated. As a result, the strain of the present invention was confirmed to have 98.39% homology with Streptomyces clavuligerus NRRL3585, a standard strain of Streptomyces clavuligerus . But, there was a little difference between the two strains in the color of medium made by a soluble pigment secreted from a strain and in the production of tacrolimus. Thus, the present inventors named the strain of the invention x Streptomyces clavuligerus CKD 1119' and then deposited it at Korean Collection for Type Cultures (KCTC) of Korea Research Institute of Bioscience and Biotechnology (KRIBB) on December 5, 2003 (Accession No: KCTC 10561BP) . The present inventors compared Streptomyces clavuligerus CKD 1119 of the invention with Streptomyces tsukubaensis No.9993 and Streptomyces sp. MA 6858, which are well-known strains to produce tacrolimus, in physiological and chemical characteristics. As a result, those three strains showed same cell wall composition that is type I and had LL type diaminopimelic acid equally, but they showed differences in cultural, morphological and physiological characteristics, in addition to the differences in the utilization of a carbon source and fermentation product. The above results indicate that the strain of the present invention belongs to the same genus where the common strains producing tacrolimus belong, but it belongs a different species from those strains.
In the present invention, a substance having immunosuppressing activity, which is produced in the strain of the invention, was separated, and then identified by nuclear magnetic resonance spectrum, infrared spectrum and high performance liquid chromatography (HPLC) . As a result, the substance having immunosuppressing activity was confirmed to be tacrolimus represented by formula 1 (see Fig. 1 ~ Fig. 4) .
The present invention also provides a method for mass-production of tacrolimus by the culture of
Streptomyces clavuligerus . The medium for the culture is preferably supplemented with malonic acid, ethanol, methionine, carbon source and nitrogen source. At this time, as a carbon source, one or more substances are preferably selected from a group consisting of starch, glucose, corn oil, glycerol, maltose, mannose and inositol, and starch, glucose and corn oil are more preferred. As a nitrogen source, one or more substances are selected preferably from a group consisting of cottonseed flour, corn steep liquor, corn steep powder, soybean flour, peptone and yeast extract, and among them, cotton seed flour and corn immersion solution are more preferred. In the preferred embodiment of the present invention, seed culture solution containing Streptomyces clavuligerus CKD 1119 was inoculated to a fermentor containing the above-mentioned medium (see Table 5) . Then, the culture solution was filtered and extraction was performed to obtain oily residue. A fraction including a target compound was purified by column chromatography, resulting in pure tacrolimus.
[Description of Drawings] Fig. 1 is a 13C nuclear magnetic resonance spectrum of tacrolimus produced in the strain of the present invention. Fig. 2 is a 1H nuclear magnetic resonance spectrum of tacrolimus produced in the strain of the present invention. Fig. 3 is an infrared spectrum of tacrolimus produced in the strain of the present invention. Fig. 4 is a high performance liquid chromatography
(HPLC) chromatogram of a sample extracted by methanol from the culture solution of the strain of the present invention.
[Mode for Invention] Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples. However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.
<Example 1> Isolation and selection of a microorganism Agar plate dilution method was used to separate a microorganism of the present invention. Particularly, 1 kg of sample soil taken from Boryeong city, Chungnam, Korea was put in a sterilized test tube and sterilized water was added thereto. After 10 minutes of settling, supernatant was obtained. The obtained supernatant was 100-fold diluted with sterilized water, and then 0.1 mi of the diluted solution was distributed onto yeast-malt extract agar (yeast extract 4 q/l , malt extract 10 q/i , glucose 4 q/l , agar 20 q/l , distilled water 1 I , pH 6.8 ~ 7.0) in petri dish. The solution was cultured at 28 °C for 21 days, after which a colony formed on the yeast-malt extract agar was picked up with a sterilized needle. The obtained colony was transferred to a fresh yeast-malt extract agar at regular interval, followed by sub-culture. The colonies proliferated during the sub-culture were further cultured at 28 °C for 10 days. To the medium, the spore suspension (1 x 107 ~ 108 cells/mβ) of Aspergillus niger ATCC 6275 was distributed by 0.5 mi . Aspergillus niger ATCC 6275 was used as a test fungus of the present invention because its' growth is inhibited by an immunosuppressant. After the distribution of the test fungus, colonies were additionally cultured for 2 more days at 28°C. Then, those colonies (276) especially inhibiting greatly the growth of Aspergilus niger ATCC 6275 were selected.
As a result, among all the selected strains, 127 strains of Streptomyces, except fungi and bacteria, were primarily selected and then among them, strains showing immunosuppressing activity were secondly selected by the below immunosuppressing activity tests.
<Example 2> Immunosuppressing activity test of the
selected strains
It was investigated whether or not the strains selected in the above Example 1 had immunosuppressing activity. Particularly, T-cells of mouse spleen were distributed into a 96-well plate for microculture containing RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with heat-inactivated 10% fetal calf serum by 200 μi per each well with the density of 0.5 x 106 cells/mi . Extraction was performed by using methanol from the culture solutions of primarily selected 127 strains, followed by concentration of the extract. The concentrated extract was distributed into wells, which were additionally supplemented with 250 nq/ i of ionomycin (a kind of ionophore, a conductor of Ca2+) and 10 nq/mi of PMA (Peptonized Milk Agar; peptonized milk 1 g, distilled water 1 I ) liquid medium. Reaction with radioisotope 3H- TdR (Thimidine deoxyribose) (S .A. (Specific activity) 5 Ci/mmol; Cone. 1 uCi/m#) was induced for about 5 hours, followed by further culture at 37 °C for 48 hours. The culture solution was filtered by glass wool to collect cells, and then radioassay was performed using a spectrophotometer . At that time, radioactivity of a control group that excluded a sample of the culture solution was regarded as 100%, providing a standard for the comparison with radioactivity of other experimental groups. For the confidence of the test results, all the above experiments were repeated three times.
As a result, only one strain showed the decrease of radioactivity under 100% in experimental groups added with the sample of the culture solution. That is, a specific factor of the strain of the present invention inhibited T- cell proliferation, and the sample of the culture solution had a factor functioning as an immunosuppressant. Therefore, the present inventors named the confirmed strain λCKD 1119' .
<Example 3> Identification of the isolated strain and
investigation of characteristics thereof
In order to identify the strain isolated in the above example 2, morphological properties of cells, cultural characteristics of cells, morphological characteristics of colonies, physiological characteristics and utilization of a carbon source were investigated.
<3-l> Morphological characteristics of cells Cell wall composition of CKD 1119 of the present invention was confirmed to be type I including LL- diaminopimelic acid and glycine. The whole cell was hydrolyzed to investigate its components, and as a result, LL-type (diaminopimelic acid was cross-link with glycine) diaminopimelic acid was observed. The results indicate that the strain has the same morphological characteristics with Streptomyces sp. strain that belongs to Actinomyces and has conidia. Therefore, the strain of the present invention has been confirmed to be a Streptomyces sp. strain.
<3-2> Cultural characteristics
Cultural characteristics of CKD 1119 isolated in the above example 2 were also investigated. Particularly, isolated strains were inoculated onto yeast-malt extract agar, potato dextrose agar, oatmeal agar, Czapek agar, glucose-asparagine agar, peptone-dextros agar and skim milk agar, followed by culturing at 28 °C for 7 - 14 days. Then, growth, spore formation, colony color and soluble pigment were investigated (Table 1)
<Table 1>
Figure imgf000016_0001
As a result, vigorous cell growth and spore formation were observed in yeast-malt extract agar medium, in which the colony color was yellow and a secreted soluble pigment was dark orange.
<3-3> Morphological characteristics of colonies
In order to investigate morphological characteristics of a novel strain of the present invention, CKD 1119 was cultured in yeast-malt extract agar at 28 °C for 14 days. Then, observation was carried out under optical and electron microscope. As a result, it was confirmed that a rectiflexible spore chain was formed and the surface of the spore was smooth. In order to investigate morphological characteristics of colonies, NRRL 3585 (ATCC 27064), a standard strain of Streptomyces clavuligerus , and a novel strain of the present invention were respectively inoculated onto yeast-malt extract agar, followed by culturing at 28 °C for 14 days. During the culture, the sizes, shapes and colors of colonies and the color change of the medium were investigated (Table 2) .
<Table 2>
Figure imgf000017_0001
As a result, the sizes, shapes and colors of colonies were looked all similar under electron microscope, and so were the morphologies of colonies. However, the color of each medium was slightly changed differently by soluble pigment secreted by the strain.
<3-4> Physiological characteristics Physiological characteristics of a novel strain of the present invention were compared with that of Streptomyces clavuligerus NRRL 3585. Particularly, the strains were cultured in a medium composed of 1 ( of distilled water, 15 g of glycerol, 38.5 g of soybean flour, 23 g of rice bran oil, 2 g of KH2P04 and 21 g of MOPS (3- [N-Morpholino] propanesulfonic acid sodium salt, CHι4N0SNa) , at 25°C for 6 days. Then, growth temperature of both strains and other physiological characteristics such as melanin pigment, starch hydrolysis, reduction of nitrogen group, gelatin liquefaction, coagulation and peptonization of litmus milk, formation of hydrogen sulfide, clavulanic acid formation and tacrolimus production, etc, were investigated (Table 3) .
<Table 3>
Figure imgf000018_0001
Figure imgf000019_0001
-; Negative response +; Positive response
As a result, the above two strains had almost same physiological characteristics except tacrolimus production, and clavulanic acid they produced was each about 50 q/ i (Table 3) .
<3-5> Utilization of carbon source Carbon source utilization of a novel strain of the present invention was compared with that of Streptomyces clavuligerus NRRL 3585.
<Table 4> Carbon source | Streptomyces clavuligerus \ CKD 1119
Figure imgf000020_0001
— ; No utilization +,-; Uncertain utilization + ; Good utilization
As a result, carbon source utilizations of the two strains were similar except glucose utilization that seemed unclear (Table 4). The above results confirmed that the CKD 1119 of the present invention was Streptomyces clavuligerus . <3-6> 16S rDNA sequence analysis
In order to identify a novel strain of the present invention more accurately, genomic DNA of the strain was separated and a whole nucleotide sequence of 16S rDNA was analyzed. Particularly, genomic DNA was separated by using the method of Rochell, et al (Rochell, P.A., et al . , FEMS Microbiol . Lett . , 100: 59-66, 1992), then PCR (polymerase chain reaction; 94 °C 1 minute, 52-56°C 1 minute, 72 °C 2 minutes) was performed to synthesize ribosomal subunit gene (16S rDNA) . Nucleotide sequence of the synthesized DNA was determined by using Tag Dye Deoxy Terminator Cycle Sequencing Kit. As a result, 1435 bp long nucleotide sequence was determined. Data base analysis with the determined nucleotide sequence was performed with NCBI (National Center for Biological Information) BLAST, confirming that the strain of the present invention showed 98.39% homology with the standard strain (NRRL 3585) of Streptomyces clavuligerus . The recent standard of determining a species is 97% homology of 16S rDNA nucleotide sequence, and based on the standard, the strain of the present invention was determined as Streptomyces clavuligerus . Thus, the present inventors named the strain of the invention λ Streptomyces clavuligerus CKD 1119' and then deposited it at Korean Collection for Type Cultures (KCTC) of Korea Research Institute of Bioscience and Biotechnology (KRIBB) on December 5, 2003 (Accession No: KCTC 10561BP) .
<Example 4> Comparison of characteristics among
Streptomyces clavuligerus CKD 1119, Streptomyces
tsukubaensis No.9993 and Streptomyces sp. MA 6858
Streptomyces tsukubaensis No.9993 and Streptomyces sp. MA 6858 are well-known tacrolimus producing strains.
The characteristics of those strains were described in US
Patent No. 5,624,842 (characteristics of Streptomyces tsukubaensis No.9993) and in US Patent No. 5,116,756 (characteristics of Streptomyces sp. MA 6858). Cell wall compositions of those three strains were all judged as type I, and LL type diaminopimelic acid was equally observed. However, other characteristics were different among them. Precisely, cultural, morphological and physiological characteristics and the carbon source utilization as well as fermentation products were all investigated and compared among them.
<4-l> Cultural characteristics Streptomyces tsukubaensis No.9993 strain secreted an orange color soluble pigment in a yeast-malt extract agar during the culture, but Streptomyces sp. MA 6858 strain did not produced any pigment. In the meantime, Streptomyces clavuligerus CKD 1119 of the present invention secreted a dark orange or red soluble pigment during the culture.
<4-2> Morphological characteristics The color of a colony was compared among the above three strains. Streptomyces tsukubaensis No.9993 strain formed a gray colony, and Streptomyces sp. MA6858 strain formed a yellowish brown or white colony. In the meantime, Streptomyces clavuligerus CKD1119 strain of the present invention formed a light yellow colony. The color of spore generated from Streptomyces sp. MA 6858 strain was light yellow or light gray, on the other hand, a spore of Streptomyces clavuligerus CKD1119 strain of the present invention showed white or gray color.
<4-3> Physiological characteristics
Starch hydrolysis was also investigated. Streptomyces tsukubaensis No.9993 was negative to the reaction but Streptomyces clavuligerus CKD1119 strain of the present invention was positive to the reaction. While Streptomyces tsukubaensis No.9993 strain showed positive response to milk peptonization, Streptomyces clavuligerus CKD1119 of the present invention was negative. Formation of hydrogen sulfide was also investigated. Streptomyces sp. MA6858 strain was positive to the formation of hydrogen sulfide, but Streptomyces clavuligerus CKD1119 of the present invention was negative to the reaction.
<4-4> Carbon source utilization
While Streptomyces tsukubaensis No.9993 strain was negative to use inositol and had a small chance of using sucrose, Streptomyces clavuligerus CKD1119 of the present invention had a small chance of using inositol and was negative to use sucrose. While Streptomyces sp. MA 6858 strain was positive to use lactose and glucose, Streptomyces clavuligerus CKD1119 of the present invention was negative to use lactose and had a just small chance of using glucose.
<4-5> Fermentation product
While Streptomyces tsukubaensis No.9993 and Streptomyces sp. MA6858 produced tacrolimus only, Streptomyces clavuligerus CKD1119 of the present invention produced not only tacrolimus but also clavulanic acid.
From the above results, it was confirmed that Streptomyces clavuligerus CKD1119 strain of the present invention is a novel strain that is different from both of the two tacrolimus producing common strains, Streptomyces tsukubaensis No.9993 and Streptomyces sp. MA6858.
<Example 5> Investigation of characteristics of an
immunosuppressant through preparation and culture of a
strain
The present inventors identified an immunosuppressant produced by CKD 1119 strain secondly selected in the above example 2. Particularly, the strain was cultured as a monocolony in a petri dish containing 30 mi of yeast-malt extract agar (starch 10 q/l , yeast extract 4 q/l , malt extract 10 q/l , agar 20 q/l , distilled water 1 , pH 6.8 - 7.0), at 28 °C for 7 - 14 days. Then, the monocolony was suspended in 1.5 mi of saline, which was distributed onto several petri dishes containing the solid medium, followed by further culture at 28 °C for 7 - 14 days. 5 mi of sterilized 15% (w/v) skim milk solution was added to each of those petri dishes, and then the dishes were scratched by incinerated spatula to obtain spore suspension. The spore suspension was passed through glass wool by a sterilized syringe, resulting in spore suspension devoid of mycelium. 100 mi Erlenmeyer flask supplemented with 20 mi of medium for seed culture represented in Table 5 was sterilized at 121°C for 20 minutes for the preparation of seed culture. The above spore suspension (2.5%(v/v)) was inoculated to the medium for seed culture, followed by shaking-culture at 28 °C with 220 rpm for 48 hours, resulting in the preparation of seed culture solution. For the main culture, 100 mi of medium for main culture represented in Table 5 was put in a 500 mi Erlenmeyer flask, which was then sterilized at 121 °C for 20 minutes. The prepared seed culture solution (10%(v/v)) was inoculated to the medium for main culture, followed by shaking-culture at 25°C with 220 rpm for 7 - 9 days, resulting in the preparation of main culture solution. The compositions of seed culture medium and main culture medium are listed in Table 5 and raw materials for industrial use with low prices were used for industrial efficiency. <Table 5>
Figure imgf000027_0001
Extraction was performed for 30 minutes from the prepared main culture solution by using 60% (v/v) methanol, followed by centrifugation at 10,000 rpm for 5 - 10 minutes. High performance liquid chromatography (HPLC) was performed with the separated supernatant under the conditions shown in Table 6, in order to investigate whether or not tacrolimus was produced.
<Table 6>
Figure imgf000028_0001
As a result, it was confirmed that the immunosuppressant produced from the strain of the present invention was tacrolimus. Major function groups of tacrolimus such as hydroxyl group (3447 cm-1) , carbonyl group (1743 cm-1) and carbon double bond (1650 cm-1) were confirmed through infrared spectrum. And 13C and 1H nuclear magnetic resonance spectrums were compared with the spectrum of standard material (US Patent No. 5,624,842), resulting in the confirmation that the immunosuppressant was tacrolimus (Fig. 1 - Fig. 4).
<Example 6> Productivity of tacrolimus through the culture of Streptomyces clavuligerus CKD1119
The tacrolimus productivity of the novel strain Streptomyces clavuligerus CKD1119' of the present invention was observed in a jar fermenter. Particularly, 5 I round flask was supplemented with 600 mi of germination culture medium, which was sterilized at 121T for 40 minutes. Then, spore suspension was inoculated thereto (final cone. 1% v/v) . The inoculated strain was shaking-cultured at 28 °C for 48 hours with 120 rpm, resulting in the preparation of germination culture solution. The germination culture solution was inoculated into a 30 I fermenter containing 18 I of germination culture medium (final cone. 3.3% v/v). Then, seed culture was performed at 28°C with 400 rpm, 1 vvm for 24 hours, which was inoculated into a 30 -β fermenter containing 18 of main culture medium (final cone. 10% v/v) , followed by further culture at 28 °C with 300 rpm, 1 vvm for 7 - 8 days . As a result, it was confirmed that 350 mg/l of tacrolimus was produced.
<Example 7> Purification of tacrolimus
Tacrolimus produced by Streptomyces clavuligerus CKD1119 of the present invention was separated by the method described in example 6 and then purified by the following method. Particularly, 18 I of the culture solution obtained in example 6 was filtered with 0.3 kg of diatomite, followed by extraction from mycelium cake with 22 I of ethyl acetate. As a result, 22 I of extract was obtained. The ethyl acetate extract obtained from the mycelium was concentrated under reduced pressure to obtain oily phase residue. The residue was mixed with acidic silica gel (special grade 12 silica gel, Fuji Devision Co.) weighing double the residue. The mixture was slurrized in ethyl acetate. A solvent was evaporated and column chromatography was performed on the same acidic silica gel (400 mi) in which dried powder was packed with n-hexane. The column was developed with n-hexane (1.2 I ) and the mixture of n-hexane and ethyl acetate (10:1 v/v, 1.2 I , 5:1 v/v, 1.2 I , 1:1 v/v, 1.2 I ^ 1:2 v/v, 1.6 ) . Fractions containing a target compound were collected and concentrated under reduced pressure, resulting in the preparation of lemon-yellow powder. Column chromatography was performed with the obtained lemon-yellow powder by following the same procedure as performed above, resulting in the preparation of 0.8 g of purified white powder type tacrolimus . [industrial Applicability] As explained hereinbefore, the microorganism of the present invention enables economical mass-production of tacrolimus, comparing to the conventional tacrolimus producing strains. And, highly qualified tacrolimus can be separated from the microorganism of the present invention, so that it can contribute to the production of an immunosuppressant which is essential for the prevention and the treatment of graft rejection accompanied with organ transplantation, graft-versus-host disease caused by bone marrow transplantation, autoimmune diseases, inflammatory diseases, etc.
Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims. SUDAΪSST TBEATY H τ wrHfitt.rrjwi. κεcoc*rπou OF xsa DEPOSIT O? MteKOOBOAKISMS ΪOR 1SE PURPOSE OΫ ΓAI-NTPSOCEDUBE INTERNATIONAL FORM RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant DO Rule 7.1
10 : KJM, YotffliHEαr. CKD Ho Cc ., $358, 3-ga, Chuπajeotjg-τp, SeodaEsπαn-fc-i, Seoul 320-756, Republic of Korea
Figure imgf000032_0001
Form BP/4 (KCTC Fwm 11) wlc juqjc

Claims

[CLAIMS]
[Claim l]
A Streptomyces clavuligerus strain producing tacrolimus represented by formula 1.
<Formula 1>
Figure imgf000033_0001
[Claim 2]
The Streptomyces clavuligerus strain as set forth in claim 1, wherein the strain is Streptomyces clavuligerus CKD 1119 (Accession No: KCTC 10561BP) .
[Claim 3] A method for mass-production of tacrolimus by culturing the strain of claim 1.
[Claim 4] The method for mass-production of tacrolimus as set forth in claim 3, wherein the culture is performed in medium containing malonic acid, ethanol, methionine, a carbon source and a nitrogen source.
[Claim 5] The method for mass-production of tacrolimus as set forth in claim 4, wherein the carbon source is selected from a group consisting of starch, glucose, corn oil, glycerol, maltose, mannose and inositol.
[Claim β] The method for mass-production of tacrolimus as set forth in claim 5, wherein the carbon source is starch, glucose or corn oil.
[Claim 7] The method for mass-production of tacrolimus as set forth in claim 4, wherein the nitrogen source is selected from a group consisting of cotton seed flour, corn steep liquor, corn steep powder, soybean flour, peptone and yeast extract.
[Claim 8] The method for mass-production of tacrolimus as set forth in claim 7, wherein the nitrogen source is cotton seed flour or corn steep liquor.
PCT/KR2004/003518 2003-12-30 2004-12-30 Microorganism producing tacrolimus and mass-productive method tacrolimus using the same WO2005063963A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020030100557A KR100485877B1 (en) 2003-12-30 2003-12-30 Microorganism producing tacrolimus and mass-productive method of tacrolimus using the same
KR10-2003-0100557 2003-12-30

Publications (1)

Publication Number Publication Date
WO2005063963A1 true WO2005063963A1 (en) 2005-07-14

Family

ID=34737934

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2004/003518 WO2005063963A1 (en) 2003-12-30 2004-12-30 Microorganism producing tacrolimus and mass-productive method tacrolimus using the same

Country Status (2)

Country Link
KR (1) KR100485877B1 (en)
WO (1) WO2005063963A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101486975B (en) * 2008-01-18 2012-04-18 浙江海正药业股份有限公司 Streptomycete, method for producing tacrolimus using the same and use thereof
US9505779B2 (en) 2010-08-24 2016-11-29 Intron Biotechnology, Inc. Tacrolimus analogues, a neuroprotective composition comprising the same, an immunosuppressive composition comprising the same, a method for preparing the same, and a mutant for producing the same

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100750810B1 (en) 2006-08-07 2007-08-22 동국제약 주식회사 Streptomyces hygroscopicus atcc 14891 mutant and production method of tacrolimus with it
KR100891313B1 (en) 2007-08-17 2009-03-31 (주) 제노텍 Method of extraction and yield-up of tricyclo compounds by adding a solid adsorbent resin as their carrier in fermentation medium
EP2272963A1 (en) 2009-07-09 2011-01-12 LEK Pharmaceuticals d.d. Process for Preparation of Tacrolimus
KR101228031B1 (en) 2009-08-25 2013-01-30 이화여자대학교 산학협력단 A method for producing FK506 from Streptomyces species
KR101197026B1 (en) 2009-11-05 2012-11-06 명지대학교 산학협력단 Tacrolimus producing strain and Tacrolimus biosynthetic genes obtained by the same

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5624842A (en) * 1984-12-03 1997-04-29 Fujisawa Pharmaceutical Co., Ltd. Strain of Streptomyces for the production of tricyclo compounds
WO1999049863A1 (en) * 1998-03-26 1999-10-07 Fujisawa Pharmaceutical Co., Ltd. Sustained release preparations
WO2003068980A2 (en) * 2002-02-13 2003-08-21 Biogal Gyogyszergyar Rt Method for extracting a macrolide from biomatter

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5624842A (en) * 1984-12-03 1997-04-29 Fujisawa Pharmaceutical Co., Ltd. Strain of Streptomyces for the production of tricyclo compounds
WO1999049863A1 (en) * 1998-03-26 1999-10-07 Fujisawa Pharmaceutical Co., Ltd. Sustained release preparations
WO2003068980A2 (en) * 2002-02-13 2003-08-21 Biogal Gyogyszergyar Rt Method for extracting a macrolide from biomatter

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101486975B (en) * 2008-01-18 2012-04-18 浙江海正药业股份有限公司 Streptomycete, method for producing tacrolimus using the same and use thereof
US9505779B2 (en) 2010-08-24 2016-11-29 Intron Biotechnology, Inc. Tacrolimus analogues, a neuroprotective composition comprising the same, an immunosuppressive composition comprising the same, a method for preparing the same, and a mutant for producing the same

Also Published As

Publication number Publication date
KR100485877B1 (en) 2005-04-28

Similar Documents

Publication Publication Date Title
JP2001226380A (en) 3-desmethylrapamycin or its derivative, and use thereof as antimycotic agent and immunosuppressant
JPH08175990A (en) Pi3 kinase-inhibiting agent and its production
KR100210482B1 (en) Streptomyces exfoliatus yj-118 and process of pravastatin sodium
JPH0763356B2 (en) New species Tolypocladium barium and method for producing cyclosporin antibiotics using the same
MXPA05005672A (en) Process for producing macrolide compound.
CN109735467A (en) A kind of streptomycete mutagenic strain of high yield tacrolimus and its application
JPH0622780A (en) Novel fungus strain and method for preparing antibiotics using same
CN105154382A (en) Gene engineering strain streptomyces tsukubaensis L20 and application thereof
WO2005063963A1 (en) Microorganism producing tacrolimus and mass-productive method tacrolimus using the same
CN108794368B (en) Alkaloid compound with diverse antibacterial activities and preparation method and application thereof
CN109182142B (en) Penicillium dermatum and application thereof
Kumar et al. Cultural, morphological and molecular characterization of vinca alkaloids producing endophytic fungus Fusarium solani isolated from Catharanthus roseus
CN109280034B (en) Benzoxazepine compound with antibacterial activity and preparation method and application thereof
CA2085522A1 (en) Microbial process for the production of trans-4-hydroxy-l-proline
CN105176904A (en) Genetic engineering strain Streptomyces tsukubaensis L21 and application thereof
CN102351859B (en) Antibiotic Pseudonocardian A and Pseudonocardian B, its preparation method thereof and its application in preparation of antibiotics and antitumor drug
CN113337432B (en) Methylophilus for producing pyrroloquinoline quinone and application thereof
EP2501821B1 (en) Process for producing primycin, primycin component(s), precursors and metabolites thereof via fementation by the use of bacterial species saccharomonospora azurea
CN110092758B (en) Novel alkaloid compound and wart spore strain for preparing compound by fermentation
US7939081B2 (en) Method for producing cercosporamide
JPH06501026A (en) Novel immunosuppressant derived from Streptomyces bregensis
EP0294491A1 (en) Antibiotic yi-hu3 and process for its preparation
CN100500668C (en) Geldanamycin derivative and its preparing method and use for preparing medicine
CN111979137B (en) Carbon-phosphorus compound derived from marine streptomyces and preparation method and application thereof
US7241588B2 (en) Method for producing novel β-lactam antibiotic from protoplast fusion strain

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

122 Ep: pct application non-entry in european phase