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WO2025177305A1 - Procédé de production d'actinomycine d - Google Patents

Procédé de production d'actinomycine d

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Publication number
WO2025177305A1
WO2025177305A1 PCT/IN2025/050245 IN2025050245W WO2025177305A1 WO 2025177305 A1 WO2025177305 A1 WO 2025177305A1 IN 2025050245 W IN2025050245 W IN 2025050245W WO 2025177305 A1 WO2025177305 A1 WO 2025177305A1
Authority
WO
WIPO (PCT)
Prior art keywords
actinomycin
purification
days
production
fermented broth
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
PCT/IN2025/050245
Other languages
English (en)
Inventor
Asha Chaubey
Neha Sharma
Ravi Singh MANHAS
Yedukondalu NALLI
Mahendra Kumar Verma
Amit Kumar
Dumbala Srinivasa REDDY
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Council of Scientific and Industrial Research CSIR
Original Assignee
Council of Scientific and Industrial Research CSIR
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 Council of Scientific and Industrial Research CSIR filed Critical Council of Scientific and Industrial Research CSIR
Publication of WO2025177305A1 publication Critical patent/WO2025177305A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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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
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
    • 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 fermentation based process for production and purification 5 of a pharmaceutically important drug (actinomycin D) using actinobacterium isolated from cold deserts of NW Himalayas. BACKGROUND OF THE INVENTION 10
  • actinomycins are a family of bicyclic chromopeptide lactones, that shares the chromophoric phenoxazinone dicarboxylic acid, to which two non-ribosomal pentapeptide lactones are attached.
  • actinomycin D The class is represented by actinomycin D, however, more than 41 actinomycins, mainly with variations in the peptide chain of the structures have been reported so far. Actinomycin was isolated for the first time by Waksman and Woodruff in 1940 from 15 Streptomyces antibioticus (Science 118, no.3062 (1953): 259-266). Although it has not been successful as an antibiotic, however, it has huge demand as a chemotherapeutic agent. Actinomycin D acts as a transcription inhibitor, which binds to the DNA duplex at the transcription initiation step, thus prevents the elongation of RNA polymerase.
  • MS449 in MPG medium (glucose 1.0 %, millet meal 2.0 %, cotton seed gluten meal 2.0 %, and MOPS 2.0 %, pH 7.0 5
  • Patent no. BR102015005953A2 has disclosed the production of actinomycin D and its analogues using Streptomyces longwoodensis with yield 1.184 g/L.
  • Medicinal Chemistry Research 17 (2008): 114-122 disclosed the production of actinomycin D with the yield of 850 mg/L using the mutant strain of Streptomyces Sindenensis.
  • CN112010942A discloses the purification of actinomycin D using ethyl acetate as solvent to obtain crude extract, followed by normal phase silica gel column, elution with cyclohexane-ethyl acetate and then dried fraction was subjected to Supercritical fluid 25 chromatography.
  • Journal of Pharmaceutical and Biomedical Analysis 195 (2021): 113835 describes the only method that denies the use of organic solvents for the purification of actinomycin D and its analogs, the method involves the use of Supercritical fluid chromatography to fractionate the 30 pure actinomycin D and its analogs.
  • Microorganisms (2020): 8(3) 351 discloses that actinomycin D could also be used as biocontrol agent against phytopathogens.
  • Table 1 Strains reported in literature for actinomycin D and their yields S.no. Strain Yield No. of References/Patent no. days 1.
  • Streptomyces griseoruber 210 mg/L 6 Praveen et.al.2009 https://doi.org/10.1111/j.147 2-765X.2009.02689.x 9.
  • Streptomyces Sindenensis-M-46 1.26 to ⁇ 2 gm/L 5 Tripathi et.al.2011 (shake flask) Journal of Chemical and Pharmaceutical Research2011, 3(5):281-289 10.
  • Streptomyces sp. MS449 (S. 1.77g/L 15 Chen et.al.2012 avermitilis) (250-ml shake https://doi.org/10.1007/s0 flask) 0253-012-4079-z 11. 467mg/L 10 Toumatia et.al.2015 Streptomyces sp. IA1 (500-ml shake flask) https://doi.org/10.1002/job m.201400202 12.
  • the yields are ⁇ two fold, which is far better than the reported literature having production of ⁇ 2.0gm/L at shake flask fermentation.
  • the present invention also involves the single step purification of Actinomycin D (>95% purity), without involving solvent extraction step with higher productivity and purity.
  • OBJECTIVES OF THE INVENTION Main objective of the present invention was to provide an improved and efficient process for the production of Actinomycin D and its analogues, an improved and efficient process for the downstream and purification of Actinomycin D and its analogues.
  • Yet another objective of the invention was to develop one step downstream processing that allows the purification of Actinomycin D.
  • the present invention relates to the field of fermentation based process development of pharmaceutically important drug (actinomycin D) using actinobacterial strain, isolated from cold deserts of NW Himalayas. PT/2025/10079
  • a fermentation process with specifically designed medium was developed for the production of actinomycin D with enhanced yields.
  • the downstream process involves purification of 5 actinomycin D by conventional solvent and single step purification method.
  • the downstream process involves purification by direct single step, which involves binding of the metabolites of fermented broth with macroporous adsorbent resin and elution of pure compound Actinomycin D using 10 methanol/water/acetone.
  • the process provided high productivity i.e. ⁇ 16.7 mg/L/h and >95% purity of actinomycin D. 15 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: A) TLC profile of Fraction.2: B) analytical HPLC analyses of Fraction 2 Figure 2: Semi-preparative Separation of Fraction 2.
  • the cultural characteristics of isolate grown on SCA were observed as powdery white colony with circular and smooth ends.
  • Molecular characterization has been done by 16S rRNA partial gene sequencing using -10 ACTT- was purified by Gel elution/SAP.
  • the purified PCR product was sequenced by Sanger sequencing herein the Sanger sequencing describes one of the DNA sequencing method by 15 Dideoxynucleotide based termination to generate family of fragments of DNA molecules which are fluorescent tagged and analyzed to generate DNA sequence and the partial sequence was assembled and submitted to NCBI with Accession number OM540372.
  • the culture has been submitted to Microbial Type Culture Collection, India with Accession no. MTCC- 25511.
  • One embodiment of the invention is the process for the production of actinomycins involves solid state as well as submerged fermentation, wherein consistent extracellular as well as intracellular production takes place in various fermentation media at shake flask level as well as in bioreactor upto 500L size, under various fermentation conditions (either alone or in 25 combination).
  • the optimization of different media herein refers to a medium for cultivation of a wide variety of microbes species including or which is liquid at room temperature.
  • Media may comprise carbon sources both simple and complex, nitrogen sources (one or multiple) along with trace elements such as Calcium carbonate, Magnesium sulphate, Di-potassium hydrogen phosphate, Ferrous sulphate, Potassium chloride, Sodium chloride, Ammonium sulphate/nitrate etc.
  • trace elements such as Calcium carbonate, Magnesium sulphate, Di-potassium hydrogen phosphate, Ferrous sulphate, Potassium chloride, Sodium chloride, Ammonium sulphate/nitrate etc.
  • the carbon sources used were selected from the list of Glucose, Fructose, Sucrose, Mannitol, Glycerol, Pectin, Lactose, Maltose, Mannose, Gluconic acid, Chitosan, Dextrin, Starch, Xylose, Molasses, Corn steep liquor, dextrin, Inositol, Chitin 5 Sorbitol where in the preferred carbon sources are glucose, starch, dextrin, chitosan, molasses, gluconic acid and glycerol either alone or combinations of these nutrients.
  • nitrogen sources used were soyabean meal, yeast extract, peptone, sodium/potassium nitrate, casein, casein 10 hydrolysate and ammonium nitrate, urea, arginine, asparagine, ammonium phosphate, ammonium sulphate, beef extract; yeast extract, soyabean meal, peptone, N/Z amine A, N/Z amine B, Casein hydrolysate, Peptone, Sodium nitrate, Valine, Urea, Arginine, Potassium nitrate, Ammonium sulphate support Actinomycin D and its analogues production; wherein preferred nitrogen sources are soybean meal, yeast extract, peptone, sodium/potassium 15 nitrate, casein, casein hydrolysate and ammonium nitrate or combinations of these nutrients.
  • the present invention involves the use of modified production medium.
  • the term production medium involves the suitable nutritional medium required by the microorganism for the production of bioactive compounds during fermentation process at bioreactor level.
  • the modified production medium involves the optimized constituent concentrations in the production medium.
  • the modified medium was designed using Design of Experiments for the production of actinomycin D and further validated by performing experiments in triplicate for better accuracy and precision.
  • Diaion HP20 column (3 cm x 60 cm, 150 mL) was prepared by soaking Diaion HP20 resin (Sigma aldrich, USA) in Milli-Q water. Finally, the column was eluted with methanol at a flow rate of 2 mL/min, collecting 30 mL fractions. Each fraction was checked for its compositions by TLC using 100% EtOAc for TLC development. Anisaldehyde stain was used for detection of the fractions and all the fractions with actinomycin D were combined and concentrated to dryness to give orange red color powder (actinomycin D). In an embodiment the purity and recovery of pure compound was compared with the conventional solvent extraction method to validate the results.
  • One embodiment of the invention is to minimize the use of solvents for the purification of 5 actinomycin D where in the fermented broth has been directly bound to macroporous adsorbent resin followed by elution step to get the purified Actinomycin D.
  • present process involves higher production of actinomycin D under optimized physico-chemical conditions along with single step downstream process for the purification, without using solvent extraction step.
  • 10 EXAMPLES The following examples are given by way of illustration of the working of the invention in actual practice and should not be construed to limit the scope of the present invention. 15
  • Example 1 Isolation and characterization of Streptomyces marokkonensis (MTCC- 25511).
  • the actinobacteria strain Streptomyces marokkonensis (MTCC-25511) was isolated from soil sample collected from cold deserts of North Western Himalayas 34.5745 0 N, 76.2320 0 E, (Barchey) India on Starch-Casein agar (SCA) medium. The pure culture was maintained and 20 preserved on same medium. The morphological characteristics were investigated on ISP2 and SCA, respectively at 28 o C ⁇ 2 o C. The cultural characteristics of isolate grown on SCA were observed as powdery white colony with circular and smooth ends. Aerial colonies appear white during early growth and yellow pigmentation starts appearing with time. 25 Molecular characterization was performed by 16S rRNA partial gene sequencing using - - ald Amp GT master mix.
  • the glycerol stocks and lyophilized cultures were kept for long term storage of the culture.
  • Table 2 Components of media for preservation of cultures Streptomyces marokkonensis MTCC-25511. Ingredients Composition (g/L) Starch 10.0 KNO 3 2.0 K2HPO4 2.0 Casein 0.3 MgSO4.7H2O 0.05 NaCl 2.0 CaCO3 0.02 FeSO 4. 7H 2 O 0.01 pH 7.0 Agar 20.0
  • Example 3 Preparation of culture inoculum Pre-inoculum was prepared in a narrow mouth 500 mL Erlenmeyer flask containing 100 mL medium (Table 3) by transferring loopful of culture from freshly grown culture plate.
  • Example 9 Effect of carbon sources on Production of actinomycin D
  • carbon sources were used to enhance the yield of actinomycin D like Glucose, Fructose, Sucrose, Mannitol, Glycerol, Pectin, Lactose, Maltose, Mannose, Gluconic acid, Chitosan, Dextrin, Starch, Xylose, Molasses, Corn steep liquor, Inositol, Chitin, Sorbitol.
  • carbon sources starch and dextrin in combination were found the main carbon sources to increase the yield of actinomycin D at maximum level.
  • Example 10 Effect of nitrogen sources on Production of actinomycin D
  • different nitrogen sources were also used to observe their effect on the yields of actinomycin D.
  • nitrogen sources like soyabean meal, yeast PT/2025/10079 extract, peptone, sodium/potassium nitrate, casein, casein hydrolysate and ammonium nitrate, urea, arginine, asparagine, ammonium phosphate, potassium nitrate, ammonium sulphate, beef extract; yeast extract, soyabean meal, peptone were found to be the suitable nitrogen sources for the maximum yield of actinomycin D and its analogues.
  • Example 11 Strain development The protoplasts were prepared from the culture of Streptomyces marokkonensis using enzymatic digestion to remove the cell wall, ensuring the formation of stable, viable protoplasts. The resulting protoplasts were then subjected to UV-induced mutagenesis under a 15 watt UV lamp for 10 minutes with a distance of 50 cm, ensuring uniform exposure to induce genetic variations.
  • Example 13 Downstream processing of fermented broth (a) Extraction, isolation, and characterization of metabolites The fermented broth (25 L) recovered after fermentation was extracted with equal volume of ethyl acetate (EtOAc) three times at room temperature. Ethyl acetate was evaporated using rotatory evaporator giving orange red colored-oily residue which on partitioning between methanol (250 mL) and hexane (250 mL) separated out the oil portion in hexane layer and a red colour solid substance in the methanol layer.
  • EtOAc ethyl acetate
  • Diaion HP20 column (3 cm x 60 cm, 150 mL) was prepared by soaking Diaion HP20 resin (Sigma Aldrich, USA) in Milli-Q water. Finally, the column was eluted with methanol at a flow rate of 2 mL/min, collecting 30 mL fractions. Each fraction was checked for its compositions by TLC using 100% EtOAc for TLC development. Anisaldehyde stain was used for detection of the fractions and all the fractions with actinomycin D were combined and concentrated to dryness to give orange red color powder (actinomycin D mg). The purity and recovery of pure compound was compared with the conventional solvent extraction method to validate the results (Table 6).
  • Present process yields (4.0 g/L) are better as compared to the conventional method used for purification of actinomycin D i.e.2.1g/L. Present process also reduces the time and solvent cost used in the conventional method. In terms of recovery 20 rate is also high i.e. >90% as compared to percent recovery in conventional method. Purity of the product in study is equivalent to the conventional method i.e >95%.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne un processus de fermentation avec un milieu spécifiquement conçu pour améliorer la production d'un médicament important sur le plan pharmaceutique (actinomycine D) en utilisant la souche actinobactérienne Streptomyces marokkonensis MTCC-25511, isolée sur les terres désertiques froides du nord-ouest de l'Himalaya (Barchey), en Inde. Le procédé comprend une fermentation de 12 jours dans des conditions de fermentation spécifiques, à savoir une température de 28 °C ± 2 °C, un pH de 7,0 ± 2,0 avec un inoculum de 10 %, suivie d'un traitement en aval faisant intervenir la purification de l'actinomycine D à l'aide d'un solvant conventionnel et d'un procédé de purification en une seule étape. La purification directe en une seule étape faisait intervenir la liaison des métabolites du bouillon fermenté avec une résine adsorbante macroporeuse et l'élution du composé pur Actinomycine D par l'utilisation de méthanol/eau/acétone. La présente invention a permis d'obtenir une productivité élevée, c'est-à-dire environ 16,7 mg/l/h d'actinomycine D avec une pureté supérieure à 95 %.
PCT/IN2025/050245 2024-02-21 2025-02-19 Procédé de production d'actinomycine d Pending WO2025177305A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN202411012579 2024-02-21
IN202411012579 2024-02-21

Publications (1)

Publication Number Publication Date
WO2025177305A1 true WO2025177305A1 (fr) 2025-08-28

Family

ID=96846580

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IN2025/050245 Pending WO2025177305A1 (fr) 2024-02-21 2025-02-19 Procédé de production d'actinomycine d

Country Status (1)

Country Link
WO (1) WO2025177305A1 (fr)

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