Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
2000-08-21
2003-03-04
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S123000
Reexamination Certificate
active
06528301
ABSTRACT:
This application is a National Stage Application of International Application Serial No. PCT/EP99/00417, filed on Jan. 21, 1999, and claims priority under 35 U.S.C. §119 to GB 9803628.8, filed on Feb. 20, 1998, and GB 9809767.8, filed on May 7, 1998.
The present invention relates to microorganisms producing a taxane, to the use of said microorganisms for the production of a taxane, and to the procedure for the isolation of said microorganisms from plants.
Paclitaxel, a diterpene which is of the chemical structural formula:
shows significant properties of promoting the polymerization of tubulin and inhibiting the depolymerization of microtubules. For these reasons, paclitaxel is a valuable antileukemic and antitumor agent and is the subject of increasing research. Paclitaxel has been identified in 1971 (Wani M C et al,
J. Amer. Chem. Soc.
1971, 93, 2325-2327) by isolating it from the bark of
Taxus brevifolia
(Pacific yew). The low yield of the isolation of paclitaxel (around 0.007%) from this source and the concerns about the survival of
Taxus brevifolia,
which is a rather slow growing plant, raised by its massive collection have pushed the research for alternative paclitaxel production methods.
Isolation of paclitaxel from a renewable source such as the needles and the twigs of Taxus species has proven possible although with a yield comparable, if not lower, than that obtainable from the bark of
Taxus brevifolia.
The total chemical synthesis of paclitaxel has been described by Nicolau et al.,
Nature
1994, 367, 630-634, nevertheless the complexity and low yield of this method have prevented its industrial scale-up.
Semisynthesis of paclitaxel from baccatin III or 10-deacetylbaccatin III, has proved to be a much better option because of the use of a renewable source such as the needles of the European yew
Taxus baccata
to obtain the precursors.
Several documents on tissue culture of plants of the genus Taxus have been published in recent years, however this procedure is limited by the intrinsic technical difficulties of cell culture on an industrial scale.
Recently, patents and patent applications for the production of paclitaxel from the fermentation of fungi and bacteria isolated from samples of Taxus have been published (WO 93/21338, U.S. Pat. No. 5,322,779, WO 95/04154, U.S. Pat. No. 5,561,055 and WO 96/32490). These confirm that there is a need for improved methods for the production of paclitaxel on industrial scale.
In this specification the used classification rules are described in Bergey's Manual of Systematic Bacteriology, vol. 4, 1989.
We have surprisingly found a new taxanes producing microorganism of the Actinomycetes group, which is particularly well suited for an industrial fermentation process for the production of a taxane such as paclitaxel and related taxanes. For instance, compared to the fungi of the prior art, the microorganisms of the present invention can be more easily genetically modified to enhance taxane production and have a shorter fermentation time.
The present invention relates to microorganisms of the Actinomycetes group which produce a taxane, such as paclitaxel or related taxanes, as well as to the biologically pure cultures of a strain of the Actinomycetes group which produce a taxane such as paclitaxel or related taxanes.
The microorganisms of the invention preferably belong to the suprageneric groups of Streptomycetes, Actinoplanetes, Maduromycetes, Thermomonosporas or Nocardioforms, more preferably they belong to the genus Streptomyces, Actinoplanes, Nocardiopsis, Micromonospora, Actinomadura or Kitasatosporia and still more preferably to the genus Kitasatosporia.
A preferred strain according to the present invention is a Kitasatosporia sp. deposited under the Budapest treaty at the Colleccion Española De Cultivos Tipo, (Biological Science Faculty, University of Valencia, 46100 Burjasot (Valencia), Spain) on Jan. 19, 1998 under the deposit number CECT 4991, and it was isolated from a
Taxus baccata
sample and proved to produce paclitaxel or other related taxanes. Therefore, the present invention more preferably relates to the biologically pure cultures of the genus Kitasatosporia having all the identifiying characteristics of Kitasatosporia sp. CECT 4991.
Moreover, the present invention provides a procedure for the isolation of microorganisms of the Actinomycetes group which produce a taxane, such as paclitaxel or related taxanes, from plants, including plants of the Taxus genus and related genera, collected from various geographical area.
Preferably, the isolation procedure of microorganisms according to the present invention is carried out on plants selected from plants of Taxus genus or related genera, more preferably
Taxus baccata, Taxus brevifolia,
or
Torreja californica.
According to the present invention, the procedure for the isolation of microorganisms of the Actinomycetes group producing a taxane, such as paclitaxel or related taxanes, may be carried out, for example, by sterilizing each part of the plant (roots, bark, twigs, needles, stems) with a sterilizing agent, preferably selected from sodium hypochlorite, propylenoxid, a solution of dodecylcarbamylmethyl-benzyldimethylammonium cloride, triethilenglycol sodium nitrite and UV light, by cutting the different parts of the plant in small fragments with a sterile blade and by placing these small fragments on agar culture media in Petri plates or, preferably, by homogenizing the sterilized parts as above, with a blender at room temperature; the resultant homogenate, reduced to powder may be then transferred on agar culture media in Petri plates using a modified Andersen's sampler. The microorganism colonies grown on the agar plates are then removed and transferred to plates containing water agar media until a pure culture is obtained.
The colonies were grown in liquid media suitable for growth and taxane production, such as conditions suitable for paclitaxel production. The extracts and the fractions obtained from the production cultures can be tested to determine the presence of taxanes, such as paclitaxel or related taxanes, using suitable assays.
In the more preferred procedure for the isolation of the microorganisms of the Actinomycetes group from Taxus and related plants, the powdered samples obtained as above described are introduced into a modified Andersen's sampler, an apparatus used to analyze the microorganisms population in air samples. Andersen's sampler is described in detail in: A. Andersen,
Journal of Bacteriology
1958, 76 (5), 471-484. This sampler is modified by associating it with a sedimentation chamber in which the powdered samples are introduced. The sedimentation chamber allows isolation of microorganisms present in low percentage from soil and vegetable powdered samples. It consists of a cylindric box made of plexiglass connected to the sampler by a rubber tubing. Plates containing different isolation media were placed in the sampler and the powdered samples were drawn. Depending on their aerodynamic dimensions, the microorganisms present in the powdered samples were collected on plates containing different isolation media. The plates are then removed, incubated and examined. The colonies of microorganisms of the Actinomycetes group grown on the agar plates were picked up and transferred on water agar media in Petri plates until a pure culture was obtained. This method enables the isolation of microorganisms of the Actinomycetes group well separated from fungi and other bacteria.
A further object of the invention is the use of said microorganisms of the Actinomycetes group for the production of a taxane, such as paclitaxel or related taxanes, which comprises the steps of
a) culturing a microorganism of the Actinomycetes group producing the taxanes in a liquid media suitable for its growth and production
b) recovering the taxanes produced.
Thus the invention provides a biologically pure culture of a microorganism of the invention. Such a culture may be substantially free of other microorganisms. The term “related tax
Breme Umberto
Caruso Marinella
Fedeli Lorena
Pavesi Andrea
Perellino Nicoletta Crespi
Marx Irene
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Pharmacia & Upjohn SpA
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