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WO1996032490A1 - Fermentation s'appliquant a la production de taxol - Google Patents

Fermentation s'appliquant a la production de taxol Download PDF

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Publication number
WO1996032490A1
WO1996032490A1 PCT/CA1996/000230 CA9600230W WO9632490A1 WO 1996032490 A1 WO1996032490 A1 WO 1996032490A1 CA 9600230 W CA9600230 W CA 9600230W WO 9632490 A1 WO9632490 A1 WO 9632490A1
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WO
WIPO (PCT)
Prior art keywords
taxol
species
microorganism
fungus
fermentation
Prior art date
Application number
PCT/CA1996/000230
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English (en)
Inventor
Jagroop S. Dahiya
Original Assignee
Novopharm Limited
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 Novopharm Limited filed Critical Novopharm Limited
Priority to AU52656/96A priority Critical patent/AU5265696A/en
Publication of WO1996032490A1 publication Critical patent/WO1996032490A1/fr

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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
    • C12P17/02—Oxygen as only ring hetero atoms
    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal 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
    • C12R2001/645—Fungi ; Processes using fungi
    • 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
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/77—Fusarium
    • 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
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/80—Penicillium

Definitions

  • This invention relates to the pharmaceutical taxol, to processes for its production, and to novel micro ⁇ organisms which can be used in fermentation process for the production of taxol.
  • Taxol is a diterpenoid amide, with significant antineoplastic properties. It shows clinical activity against ovarian and breast cancer. It has the following chemical structural formula:
  • the present invention provides taxol-producing microorganisms derived from Taxus hicksii. According to another aspect, the invention provides a process for preparing taxol which comprises fermenting an appropriate substrate with a microorganism derived from Taxus hicksii.
  • the present invention provides a process for obtaining a microorganism capable of producing taxol on fermentation of an appropriate substrate therewith, which comprises extracting the microorganism from the leaves, twigs and/or bark of the ornamental yew shrub Taxus hicksii.
  • FIGURES 1 and 2 are HPLC traces derived from products of Example 10 below,-
  • FIGURE 3 is a copy of the UV spectrum of the taxol sample produced according to Example 11 below;
  • FIGURE 3A is the corresponding UV spectrum for standard taxol from the prior art
  • FIGURES 4A, 4B and 4C are NMR spectra of taxol samples from Example 11 below and the prior art.
  • Taxus hicksii is an ornamental shrub, member of the yew family, which is readily available from garden stores in North America, and is commonly cultivated in gardens, for ornamental purposes. It is quite distinct from the Pacific yews (Taxus brevifolia) from which taxol has previously been extracted as a bark component. Taxus hicksii grows in temperate climates, to a maximum height of about 4-5 feet. A common, colloquial name for the plant is yew hicks. Microorganisms capable of producing taxol are extracted from the plant by leaching appropriate parts of the plant with an inert solvent such as water.
  • an inert solvent such as water.
  • Appropriate parts of the plant include the needles (leaves) , twigs, branches and bark of the plant. These parts may be cut from the plant and homogenized, e.g. with distilled water under agitation in a standard homogenizer. The microorganisms, presumably deriving from the plant spores, are found in the liquid phase from the homogenate.
  • the preferred parts of the plant are the leaves (needles) , especially the leaves exhibiting black spot discoloration, since these show the clearest evidence of the likely presence of suitable microorganisms. They may be isolated from the liquid phase of the homogenization by filtering off the solid debris, and plating the filtrate onto appropriate plates containing nutrient medium for microbial growt .
  • Such growth reveals the presence of at least three different types of microorganism, which can be identified visually by microscopic examination.
  • the three types are bacteria, showing mostly gram +ve rods; Actinomycetes,- and fungi. Both the Actinomycetes and the fungi derived from Taxus hicksii and their use in producing taxol are embraced by the scope of the present invention, with the fungal microorganisms constituting the preferred embodiment, on account of the yields of taxol obtainable by use of them.
  • Taxol is not the type of product to be producible using bacteria.
  • an appropriate fungal strain When an appropriate fungal strain has been identified and isolated, it is used in fermentation procedures for the preparation of taxol.
  • the fungus is first initiated in a nutrient seed medium containing an appropriate carbon source such as a sugar, and conventional nutrients, minerals, vitamin additives and the like.
  • an appropriate carbon source such as a sugar, and conventional nutrients, minerals, vitamin additives and the like.
  • the seed culture containing it may be transferred to a production medium broth containing the appropriate sources of carbon, hydrogen, oxygen and nitrogen, along with various vitamins, mineral salts nutrients, etc., to promote the growth of the fungus and the production of the desired end product.
  • the seed medium and the production medium both contain standard ingredients known to those skilled in the art for the cultivation of fungal microorganisms. Specific examples of appropriate such media are given below in connection with the specific working examples. Room temperature, aerobic cultivation is generally adopted.
  • the taxol product is produced inside the fungal cells, so that it is found in the solid or semi-solid portion of the production medium, i.e. in the biomass. It may be extracted therefrom by acidifying the fermented broth and filtering it to obtain the fungal cake therefrom.
  • the fungal cake can be treated with a suitable solvent, e.g. ethyl acetate, to effect solvent extraction of the taxol. It can be purified by further solvent extractions, filtrations, chromatography and the like. Taxol purities in excess of 80% are readily obtained.
  • Yew tree branches, twigs and needles of a Taxus hicksii ornamental yew shrub were obtained from North York, Ontario. These samples (106 g) were homogenized in sterile distilled water, using a high shear agitation homogenizer, laboratory scale. After homogenization at room temperature, the mixture was filtered, and the filtrate (0.1 mL) was plated onto potato-dextrose agar (PDA) plates (6x) . These PDA plates, after pour plating, were incubated at 28°C for six days. After six days, the plates were examined for micro-organisms. Three types of micro ⁇ organisms were found on the plates, namely bacteria (mostly gram +ve rods), actinomycetes and fungi.
  • the fungi were initially examined for taxol- producing properties on fermentation, and four separate isolates of fungi were obtained and studied. The fungal colonies were picked at random, purified by subculturing on PDA media plates. After purification, the fungal isolates were identified by morphological studies.
  • a colony on PDA medium plate is light yellow to deep orange in colour, densely coloured at the center, slow-growing, reaching 34 mm in diameter in 10-12 days at 25°C.
  • the colony surface is merishmoid and wrinkled to even slimy.
  • Aerial mycelia are rare to absent.
  • Conidia are borne directly from hyphae or from denticles formed laterally on hyphae. When the denticle elongates it is septated at the base and regarded as a phialide, which is rarely observed.
  • Phialides are subulate, borne singly, or in groups, simple or branched monophialides. Conidia show wide variation in shape, they are thin walled, generally crescent to thread-like with pointed apical cells and indistinctly or apedicellate foot cells to small and strongly curved. The conidia are mostly 30-60 ⁇ m x 2-3 ⁇ m in size. Chlamydospores are borne terminally or intercalary formed singly or in chains, globose, smooth-walled 4-7 ⁇ m in diameter. Based on these morphological features, this fungus was tentatively identified as species of the genus Fusarium belonging to the order Fungi imperfecti . ISOLATE #2
  • Metulae are cylindrical 8-12 ⁇ m long. Phialides are ampulliform, 6-12 ⁇ m. Conidia are spherical to sub-spherical, 2-4 ⁇ m with smooth to finely roughened wall, borne on phialides in divergent and disordered chains. This fungal isolate was identified as a species of the genus Penicillium belonging to the order Funcri imperfecti based on the morphological features.
  • Conidia less than 0.3 mm in diameter, small, dark greyish areas embedded among stromatic growth, exuding pale, greyish yellow conidial masses.
  • Conidiomata are pycnidia, 250-400 ⁇ m in diameter, sub-globose, translucent to dark greenish grey, densely gregarious to confluent, exuding pale, greyish yellow conidial masses, ostiolate, covered with white mycelium when young, glabrous in age with walls composed of pseudo-parenchymatous tissue, 2-4 cell layer thick, with internal cavity lined with conidiogenou ⁇ cells.
  • Conidiogenou ⁇ cells are enteroblastic, phialidic, 4-6 ⁇ m in diameter, pyriform, sub- globose to doliform with 2-4 conidiogenous loci.
  • Conidia aseptate hyaline, smooth-walled 1.5-2.5 x 34 ⁇ m. Based on morphological features this fungal isolate was identified as a lingham species of the genus Pho a . belonging to the order Fungi Imperfecti .
  • This fungus grows readily and sporulates abundantly with little or no nonsporulating aerial mycelium.
  • individual erect conidiophores rise directly from the agar substrate and bear terminal clumps of conspicuously branched chains of conidia, approximately 50-70 conidia constitute the 4-8 branches of a fully developed head.
  • the initial conidia produced directly from the conidiophore or within one to two conidia above it tend to be ellipsoid, 5-7 transversely septate and about 30-40 x 8-12 ⁇ m, as the branching chains elongate, the more recently formed conidia are ellipsoid to ovoid, 3-5 transversely septate and about 12-20 x 8-10 ⁇ m.
  • Juvenile conidia of the initial sporulation are narrowly ovoid with densely minutely granulate walls. This dense ornamentation persists throughout the enlargement of these initial conidia to such an extent that when viewed in mounts under 100 x magnification, their internal septa are obscured and the septation pattern indistinct. This punctuate ornamentation, the usual condition, sometimes may develop into coarser varicosity as conidia age. Juvenile conidia produced later in the chains are ovoid and also granulate, but not so much so that septa are obscured. Secondary conidiophores at the apex of conidia usually are l-celled (2-4 ⁇ m, long) uncommonly extended geniculately to 15-20 ⁇ m.
  • this isolate was identified as altemata species of the genus Altemaria. belonging to the order Fungi Imperfecti.
  • the sub-species isolated was named from its origin, i.e. Altemariaretemata (Taxus hicksii) , and code named NBTX-JA-1. Viable samples thereof have been deposited with International Mycological Institute, Egham, Surrey, England under numbers IMI 360997a and IMI 360997b. Viable samples thereof have also been deposited on April 7, 1995, with American Type Culture Collection, under No. 74335.
  • a frozen vial of NBTX-JA-1 was transferred aseptically into seed media (50 ml) in an Erlenmyer flask (250 ml capacity) and incubated on a shaker (New Brunswick) at 28°C and 200 rpm for 48 hours.
  • seed media had the following composition:
  • seed culture (10 mL) was inoculated into a production medium, the composition of which varied from example to example, contained in a flask (2L capacity) .
  • the medium was incubated at 28°C on a shaker (200 rpm) for 12 days.
  • Each medium contained 1 Litre of tap water, and had a starting pH of 6.0.
  • the fermented broth in each case was extracted and assayed for taxol content. Extraction was accomplished by homogenizing, for 3 minutes, and acidifying a fermented broth sample (20 ml) to pH 4.0 with 4N-HC1. An equal volume of ethyl acetate (20 ml) was added to the acidified mixture in a conical, disposable plastic centrifuge tube. The tube and its contents were shaken overnight at room temperature. The next day it was centrifuged at 2500 g for 10 minutes. The ethyl acetate layer was removed and 10 mL thereof dried completely by evaporation in vacuo. The residue was dissolved in methanol (10 mL) .
  • the crude fermented broth extracts in methanol so obtained were subjected to HPLC analysis. They were analyzed on reversed-phase C l ⁇ column (Curosil-A, 25 x 0.46 cm; I.D. Phenomenex, Torrance, California, U.S.A.) using a mobile phase of acetonitrile : 0.01 M phosphoric acid (65:35 v/v) and a flow rate of 1 mL/min.
  • the UV detector (Shimadzu SPD-10 AV) was set at ⁇ 230 nm. Throughout the experiments, all injection volumes were 20 ⁇ l. All samples were filtered through 0.45 ⁇ m filter prior to HPLC analyses. Quantitation of taxol in crude samples was done by comparing with a known concentration of standard taxol purchased from Sigma Chemical Co., St. Louis, MO, USA.
  • the mycelial and agar bits were stirred vigorously for 2-3 minutes.
  • the seed culture (250 mL) was transferred aseptically to a Chemap Fermenter (CBC-10) containing sterile production medium (14L) , of the following composition, in g/L:
  • the culture was allowed to ferment up to 114 hours with pH maintained between 6.4-6.8 during fermentation with lON-NaOH.
  • the dissolved oxygen (D.0%) was varied between 35-100%.
  • Fermented broth samples were withdrawn from the fermenter at different time intervals during the course of fermentation and extracted with ethyl acetate and assayed for taxol titre of analytical HPLC.
  • the samples were replaced with an equal amount of feed medium, of the following composition, in g/L: Glucose 100
  • Fig. 1 of the accompanying drawings is the HPLC trace of the ethyl acetate extract
  • Fig. 2 is that of the methanol extract.
  • methanol extracted more taxol followed by dichloromethane these solvents extracted more impurities. Accordingly, ethyl acetate is the preferred extraction solvent.
  • the purified taxol compound prepared by fermentation of NBTX-JA-1 as described in the previous examples was subjected to UV spectral analysis, FAB-MS spectral analysis and 1 HNMR spectral analysis, and compared with the respective spectra of authentic taxol standard purchased from Sigma Chemical Co., St. Louis, MO, USA. In each case, the identity of the compound produced according to the present invention was confirmed.
  • Figures 4A, 4B and 4C are comparative - ⁇ NMR analyses of compound purified from fermented broth and standard taxol sample.
  • This crude taxol (328.20 mg) was purified by dissolving in methanol (100 mL) and heating slowly up to boiling on a water bath for 10 minutes. The flask containing the solution was then immediately transferred to a cold cabinet at 10 * C and allowed to cool overnight. The white mealy coloured precipitates obtained were filtered through a Whatman #1 filter paper. The precipitates were dried in vacuo at 40°C. The precipitates (278.5 mg) were repurified as described above, and checked for purity by HPLC analysis.

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Abstract

Des micro-organismes produisant du taxol sont isolés des feuilles, des branches, des ramilles et de l'écorce du bois d'if ornemental Taxus hicksii et utilisés dans un processus de fermentation pour produire une biomasse contenant du taxol dans des quantités relativement importantes. Un de ces micro-organismes spécifiques produisant du taxol a été identifié comme étant un champignon de l'espèce Alternaria alternata, de la famille Fungus imperfecti, et référencé dans cette demande sous le nom d'Alternaria alternata (Taxus hicksii).
PCT/CA1996/000230 1995-04-14 1996-04-12 Fermentation s'appliquant a la production de taxol WO1996032490A1 (fr)

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AU52656/96A AU5265696A (en) 1995-04-14 1996-04-12 Fermentation for taxol production

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US08/422,410 1995-04-14

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999032651A1 (fr) * 1997-12-22 1999-07-01 Bcm Developpement Inc. Production de masse de taxanes et de paclitaxel a l'aide de bacteries
WO1999042561A1 (fr) * 1998-02-20 1999-08-26 Pharmacia & Upjohn S.P.A. Production de paclitaxel par actinomycetes
WO2006009518A1 (fr) * 2004-07-19 2006-01-26 Haiyi Biotech Pte Ltd Micro-organisme et procede de fabrication de paclitaxel au moyen de ce micro-organisme
WO2006036131A1 (fr) * 2004-09-29 2006-04-06 Haiyi Biotech Pte Ltd Nouveau compose et nouveau micro-organisme permettant de le produire
CN100420679C (zh) * 2004-09-29 2008-09-24 汕头市双骏生物科技有限公司 化合物、菌株、及应用该菌株生产该化合物的方法
WO2014090208A2 (fr) 2012-12-14 2014-06-19 Forschungszentrum Jülich GmbH Procédé d'identification d'une cellule présentant, comparativement à son type sauvage, une concentration intracellulaire augmentée d'un métabolite déterminé, la modification de la cellule s'effectuant par recombinaison, un procédé pour produire une cellule de production d'un métabolite déterminé, génétiquement modifiée comparativement à son type sauvage, ladite cellule étant à production optimisée, un procédé pour produire ledit métabolite ainsi que des acides nucléiques requis à cet effet

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993021338A1 (fr) * 1992-04-16 1993-10-28 The Research And Development Institute, Inc. Production de taxol par un microbe

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993021338A1 (fr) * 1992-04-16 1993-10-28 The Research And Development Institute, Inc. Production de taxol par un microbe

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999032651A1 (fr) * 1997-12-22 1999-07-01 Bcm Developpement Inc. Production de masse de taxanes et de paclitaxel a l'aide de bacteries
WO1999042561A1 (fr) * 1998-02-20 1999-08-26 Pharmacia & Upjohn S.P.A. Production de paclitaxel par actinomycetes
US6528301B1 (en) * 1998-02-20 2003-03-04 Pharmacia & Upjohn Spa Paclitaxel production by actinomycetes
WO2006009518A1 (fr) * 2004-07-19 2006-01-26 Haiyi Biotech Pte Ltd Micro-organisme et procede de fabrication de paclitaxel au moyen de ce micro-organisme
WO2006036131A1 (fr) * 2004-09-29 2006-04-06 Haiyi Biotech Pte Ltd Nouveau compose et nouveau micro-organisme permettant de le produire
CN100420679C (zh) * 2004-09-29 2008-09-24 汕头市双骏生物科技有限公司 化合物、菌株、及应用该菌株生产该化合物的方法
WO2014090208A2 (fr) 2012-12-14 2014-06-19 Forschungszentrum Jülich GmbH Procédé d'identification d'une cellule présentant, comparativement à son type sauvage, une concentration intracellulaire augmentée d'un métabolite déterminé, la modification de la cellule s'effectuant par recombinaison, un procédé pour produire une cellule de production d'un métabolite déterminé, génétiquement modifiée comparativement à son type sauvage, ladite cellule étant à production optimisée, un procédé pour produire ledit métabolite ainsi que des acides nucléiques requis à cet effet
DE102012024435A1 (de) 2012-12-14 2014-07-10 Forschungszentrum Jülich GmbH Verfahren zur Identifizierung einer Zelle mit gegenüber ihrem Wildtyp erhöhten intrazellulären Konzentration eines bestimmten Metaboliten, wobei die Veränderung der Zelle durch Rekombi-neering erreicht wird, sowie ein Verfahren zur Herstellung einer gegenüber ihrem Wildtyp genetisch veränderten Produktionszelle mit optimierter Produktion eines bestimmten Metaboliten, ein Verfahren zur Herstellung dieses Metaboliten, sowie dafür geeignete Nukleinsäuren

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