WO1996032490A1

WO1996032490A1 - Fermentation for taxol production

Info

Publication number: WO1996032490A1
Application number: PCT/CA1996/000230
Authority: WO
Inventors: Jagroop S. Dahiya
Original assignee: Novopharm Limited
Priority date: 1995-04-14
Filing date: 1996-04-12
Publication date: 1996-10-17
Also published as: AU5265696A

Abstract

Taxol producing microorganisms are isolated from the leaves, branches, twigs and bark of the ornamental yew shrub Taxus hicksii, and used in fermentation process to produce a biomass containing taxol in relatively large quantities. One specific such taxol-producing microorganism has been identified as a fungus of the species Alternaria alternata, of the order Fungus imperfecti, and herein designated Alternaria alternata (Taxus hicksii).

Description

FERMENTATION FOR TAXOL PRODUCTION

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:

It appears to act by promoting the polymerization of tubulin and inhibiting the depolymerization of microtubules.

One serious obstacle to the development of taxol into a readily prescribable and available anti-cancer drug is its supply. It was originally isolated from the bark of the Pacific yew (Taxus brevifoli) . but it is present therein in only very small amounts (about 0.007%). The collection of large amounts of bark just for clinical trials has raised concerns about the impact of continued collection on the survival of _£__. brevifola. Whilst collection procedures have improved to the point where the amount of taxol from bark has doubled on a percentage basis, and other Taxus species have been found to contain

SUBSTITUTE similarly small amounts of taxol in their leaves (needles) , sources other than natural sources need to be developed if taxol is to meet its potential demand at reasonable prices.

Chemical processes for preparing taxol have been described in the prior patent literature. Thus Canadian Patent 1.324.611 C.N.R.S describes a chemical process for preparing taxol from a taxane compound having a free hydroxyl group at position 9. This and similar processes still rely on plant-derived starting material, and do not satisfactorily overcome the supply problems.

Canadian patent application 2.080.000 Nippon Steel Corporation describes a tissue culture procedure for making taxol, using plant cells of the Taxus genus.

International Patent Application PCT/US93/03416 Strobel et al describes a process for preparing taxol microbiologically, by fermentation using a fungus derived from the bark of Taxus brevifolia. and presumably that responsible for the natural production of taxol in Taxus brevifolia. The inventors name the fungal species Taxomyces andreae. However, they report the production of taxol using such a fungus in culture broths at a yield of only about 24-50 nanograms per litre (a nanogram is 1/1,000,000,000 grams).

It is an object of the present invention to provide a novel process for the production of taxol.

It is a further object of the invention to provide novel microorganisms which, on fermentation, will produce taxol in yields significantly higher than those hitherto reported for in vitro taxol producing processes.

It has now been discovered that the ornamental yew shrub having the botanical name Taxus hicksii contains microorganisms capable of fermentation to produce relatively high yields of taxol. Accordingly, the present invention, from one aspect, 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. According to a further aspect, 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.

The plant 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. 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.

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. Once growth of the microorganism is properly established and underway, 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. In general terms, 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.

The invention will be further described with reference to the following specific examples.

EXAMPLE 1 - ISOLATION OF MICRO-ORGANISMS

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.

ISOLATE #1

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. There is a tendency for hyphae on the surface of agar and aerial mycelium to form mycelial ropes with several strands. 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 30-60μm x 2-3μm in size. Chlamydospores are borne terminally or intercalary formed singly or in chains, globose, smooth-walled 4-7μm 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

Colony on Czapek dox agar plates 15-16 mm in diameter at 25°C after 7 days of growth. Thick and dense hyphal mat is formed and the surface is downy. The colony is slightly umbonate at the center, and radially sulcate. Greyish green conidia are formed at the center. Hyaline to golden yellow exudate is produced. No water soluble pigments are produced. The reverse side of the colony is yellowish brown, and radially and concentrically sulcate. Colony on malt extract peptone agar is 15-16 mm in diameter at 25°C after 6-7 days of growth. Penicilli are terverticillate, partly biverticillate. The conidiophore wall is smooth, or very finely roughened. Metulae are cylindrical 8-12μm long. Phialides are ampulliform, 6-12 μm. Conidia are spherical to sub-spherical, 2-4μm 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.

ISOLATE #3

Colonies on Czapek dox agar after 2-3 weeks at 25°C, plain at margin, with irregular εtromatic growth radiating from inoculation point, with cottony white, aerial mycelium on surface of stroma, but aerial mycelium sparse and floccose or appressed toward margin, pale olive grey. 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 μm 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 conidiogenouε cells. Conidiogenouε cells are enteroblastic, phialidic, 4-6μm in diameter, pyriform, sub- globose to doliform with 2-4 conidiogenous loci. Conidia aseptate hyaline, smooth-walled 1.5-2.5 x 34 μm. Based on morphological features this fungal isolate was identified as a lingham species of the genus Pho a . belonging to the order Fungi Imperfecti .

ISOLATE #4

This fungus grows readily and sporulates abundantly with little or no nonsporulating aerial mycelium. Typically, 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 μm, 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 μm. 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 μm, long) uncommonly extended geniculately to 15-20 μm. Based on morphological features 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. Altemaria altemata (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.

EXAMPLE 2-8 - TAXOL PRODUCTION IN SHAKE FLASK EXPERIMENT

A series of seven experiments was conducted to product taxol by fermentation of the fungal isolate NBTX- JA-l in different media, using the following procedure.

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. In each case the seed media had the following composition:

glucose 10 g/L

Pharmamedia 20 g/L

Tap water IL pH 6.8

At the end of incubation, 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 various media formulation are given below in Table 1. Unless otherwise stated, the amounts are given in g/L.

Triplicate runs of each formulation were conducted.

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_lβ 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 λ230 nm. Throughout the experiments, all injection volumes were 20 μl. All samples were filtered through 0.45 μm 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 results are shown below in Table 2. TABLE 2

Example # Final pH PMV(%) Taxol Titre

(μg/ml)

2 (i) 5.28 30 484

(ϋ) 5.08 35 446

(iii) 5.16 32 469

3 (i) 4.98 35 459

(ii) 5.02 40 450

(iii) 5.12 40 512

4 (i) 5.23 30 409

(ii) 5.41 25 361

(iii) 5.28 30 357

5 (i) 5.61 35 400

(ii) 5.29 25 364

(iii) 5.36 30 362

6 (i) 5.98 40 393

(ii) 5.72 45 361

(iii) 5.66 50 374

7 (i) 5.62 45 364

(ii) 5.78 50 332

(iii) 5.68 35 337

8 (i) 5.36 40 358

(ii) 5.44 45 330

(iii) 5.47 40 334

EXAMPLE 9 TAXOL PRODUCTION IN 20L FERMENTER

SEED INOCULUM PREPARATION

A PDA slant culture of NBTX-JA-1 (8 days old growth) was used for seed inoculum preparation. A sterile solution of Triton-X-100 (0.001 %, v/v) (10 mL) was added aseptically to the PDA slant culture of NBTX-JA-1. The mycelium and the conidia were scraped with a sterile glass rod. The mycelial and agar bits were stirred vigorously for 2-3 minutes. The mycelial suspension (10 mL) was transferred aseptically to an Erlenmeyer flask (2 L capacity) containing autoclaved seed medium (500 mL) . After inoculation, the seed media flask was incubated on a New Brunswick shaker (200 rpm) at 28°C for 48 hours. The seed medium had the following composition, in g/L:

Glucose 10

Pharmamedia 20

Tap Water 1 L

PH 6.8

PRODUCTION STAGE

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:

Glucose 60

Malt Extract 10

Corn Steep Solid 2

Peptone 1

Yeast Extract 2

CaCl- 2

KH₂P0₄ 1

(NH₄)₂S0₄ 1

ZnS0₄.7H-0 0.05

MnS0₄.4H-0 0.05

Sodium Benzoate 0.05

pH 6.8

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

Malt Extract 10

Corn Steep Solid 2

Peptone 1

Yeast Extract 2

Tap Water 1 L pH 6.5

The extraction and preparation of samples for HPLC analysis was conducted as described in Examples 2-8. The results are shown below in Table 3.

TABLE 3

TAXOL PRODUCTION I 20 L FERMENTER (14 L WORKING VOLUME)

Time Final pH PMV(%) TAXOL (μg/mL)

0 6.79 1.5 0

41.5 6.42 50 0

56 6.58 50 0

66 6.59 52 104 . 5

74 6.60 55 144 . 6

89.5 6.67 52 230 . 7

97 6.69 50 243 . 8

114 6.80 48 237 . 8

EXAMPLE 10 - EFFECT OF DIFFERENT SOLVENTS ON

EXTRACTION OF TAXOL FROM FERMENTED BROTH OF NBTX-JA

An aliquot of fermented broth (2L) prepared according to the previous Example but on a larger scale was acidified with ION HCI to pH 4.0 and filtered through Whatman #1 filter paper. The fungal cake was retained (40.8 g) was retained. The fungal cake (5 g/flask) was distributed into Ehrlenmeyer flasks (250 mL capacity) and the cake was homogenized with different solvents (ethyl acetate, methanol, ethanol, acetone, hexane and methylene chloride, 50 mL of solvent per flask. Samples (5 mL) from each treatment were withdrawn and dried in vacuo at 40"C. Each dried residue was reconstituted in methanol (5mL) and analyzed for taxol content by HPLC as previously described. The results are given below in Table 4.

TABLE 4 EFFECT OF DIFFERENT SOLVENTS ON EXTRACTION

OF TAXOL FROM FERMENTED CAKE

Solvent Amount of cake used Amount of taxol in crude

(g) (mg/g of cake)

Acetone 5.0 1.718

Ethanol 5.0 1.384

Methanol 5.0 2.316

Ethyl acetate 5.0 1.519 H Heexxaannee 5 5..00 1.270

Dichloromethane 5.0 2.174

Fig. 1 of the accompanying drawings is the HPLC trace of the ethyl acetate extract, and Fig. 2 is that of the methanol extract. Although methanol extracted more taxol followed by dichloromethane, these solvents extracted more impurities. Accordingly, ethyl acetate is the preferred extraction solvent.

EXAMPLE 11 - IDENTIFICATION BY SPECTRAL ANALYSIS

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 ¹HNMR 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.

Thus for UV spectral analysis, the purified compound (1 mg) was dissolved in methanol (100 mL) and subjected to UV spectral analysis on a Shimadzu UV - visible spectrophotometer (UV-160) using methanol as reference solvent. Figure 3 of the accompanying drawings is a copy of the UV spectrum so obtained, and Figure 3A is the corresponding UV spectrum of the standard taxol.

The following Table 5 gives the NMR analyses data for the compound prepared according to the invention, with the corresponding data for standard taxol in parentheses.

TABLE 5 -ΗNMR Analysis (300 MHz; CDCL₃) of Purified Compound

Values inside parentheses are for standard taxol. Figures 4A, 4B and 4C are comparative -ΗNMR analyses of compound purified from fermented broth and standard taxol sample.

Based on UV, HPLC, FAB-MS and ¹HNMR analyses the compound purified from fermented broth of NBTX-JA-l was identified as taxol.

EXAMPLE 12 - EXTRACTION OF TAXOL FROM FERMENTED BROTH WITH ETHYL ACETATE

2.4 L of fermented broth from a fermentation using NBTX-JA-I and conducted as previously described was filtered through a Whatman #1 filter on a Buchner funnel under vacuum. 87.56g of wet cake was collected. The wet cake was homogenized in ethyl acetate (500 ml. pH 4.0) for five minutes and filtered. The cake was then rehomogenized in a fresh lot of ethyl acetate (500 mL) . The ethyl acetate extract after filtration was evaporated to dryness and the residue (876 mg) was redissolved in methanol (100 mL) and assayed for taxol content in the cake by HPLC analysis (dil. factor = 9) .

By calculation of peak areas due to taxol presence and comparison with results from standard taxol solutions, it was determined that the taxol content of the crude sample injected was 364.67 μg/ml, and that the total amount of taxol (in crude form) in the 87.56 g of fungal cake was 328.20 mg.

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.

Claims

WHAT IS Cτ**T TϋT> TS :

1. A process for producing taxol, which comprises fermenting an appropriate substrate with at least one microorganism obtained from the plant species Taxus hicksii to obtain a taxol-containing biomass, and recovering the taxol from the biomass so formed.

2. The process of claim 1, wherein the microorganism is a fungus of a species of the genus Fusarium. belonging to the order Fungi imperfecti.

3. The process of claim 1, wherein the microorganism is a fungus of the species lincrham. of the genus Phoma. belonging to the order Fungi imperfecti.

4. The process of claim 1, wherein the microorganism is a fungus of a species of the Penicillium genus, belonging to the order Fungi imperfecti.

5. The process of claim 1, wherein the microorganism is a fungus of the species altemata. genus Altemaria. sub-species Taxus hicksii. code referenced herein NBTX-JA- I.

6. The process of claim 1, wherein the taxol is recovered from the biomass by dissolution of the taxol in a low molecular weight organic solvent.

7. The process of claim 6, wherein fermentation broth is acidified and filered to obtain a fungal cake containing taxol, and the fungal cake is subjected to solvent extraction.

8. The process of claim 7, wherein the solvent is selected from the group consisting of acetone, ethanol, methanol, ethyl acetate, hexane and dichloromethane.

9. The process of claim 8, wherein the solvent is ethyl acetate.

10. Microorganisms capable of producing a taxol- containing biomass upon fermentation in an aqueous fermentation broth containing appropriate sources of carbon, hydrogen, nitrogen and fermentation nutrients, said microorganisms having been obtained by solvent leaching of appropriate parts of the plant species Taxus hicksii.

11. A microorganism according to claim 10 which is a fungus of a species of the genus Fusarium. belonging to the order Fungi imperfecti.

12. A microorganism according to claim 10 which is a fungus of the species lingham. of the genus Phoma. belonging to the order Fungi imperfecti.

13. The fungal microorganism Altemaria altemata (Taxus hicksii) .

14. A process of obtaining microorganisms capable of producing taxol on fermentation, which comprises leaching appropriate parts of the plant Taxus hicksii with an aqueous solvent, treating the resulting aqueous liquid phase to recover microorganisms therefrom, cultivating the organisms so obtained and selecting those capable of producing taxol on fermentation.

15. The process of claim 14, wherein the plant parts are the leaves, twigs, branches or bark.

16. The process of claim 15, wherein the plant parts are leaves showing black spot discoloration.

17. The process of claim 15, wherein the plant parts are homogenized with the aqueous solvent, followed by separation of the solids residue from the microorganism- containing liquid.