Plant Cell Cultures

Technical Field of the Invention

The field of this invention is a technique for the screening of cell lines for phytochemical production by plant cell culture. More particularly, the present invention pertains to the screening of liquid suspension cell lines that can secrete the plant chemical into a second phase present along with the medium and cellular phase. The technique can also be used for production of phytochemicals from cultured plant cells.

Background of the Invention

Present techniques for producing phytochemicals and for screening cell lines to identify phytochemical producers rely on the characterization of cell lines through repeated-subculture on semi-solid medium or in suspension, where other process enhancement strategies are ignored. Once a high yielding cell line is found, process enhancement strategies are then applied and the process conditions optimized again. This has the disadvantage that cell lines more amenable to process enhancement strategies, such as two-phase culture, may be ignored in the primary screening. In classical screening methods, the optimization of culture condition is a two-step process; once during screening and then under processing conditions. Furthermore, in the classical method, the cells are sacrificed to detect the level of intracellular phytochemical.

Various metabolic control mechanisms prevent cells from unnecessary metabolite production. This is contrary to the whole purpose of phytochemical production by plant cell culture, which is the application of cultured plant cells to the production of valuable

secondary metabolites. To solve this from a process engineering viewpoint, removal of the product from the reaction site by integration of a primary down-stream processing step is one of the possible strategies.

The major incentive towards integrated fermentation and separation in plant cell culture deals with avoiding feedback repression and product degradation. Feedback repression comprises a group of control mechanisms in a cell that regulate the production capacity of the cell by adapting the corresponding enzyme activities in response to high product concentrations. High product concentrations therefore reduce the maximum production capacity of the cell for the particular product. Feedback repression especially occurs during the production of secondary metabolites because growth and production are not directly coupled. If product concentrations are too high, they result in decreased product formation capacity when compared with biomass growth and thus in a low metabolite yield with respect to substrate or precursor. Also avoiding product degradation during fermentation in plant cell cultures plays a key role in using integrated processes. Plant cell fermentation usually lasts for a couple of weeks and during this time frame extracellular enzymes or light coupled reactions may degrade the secondary metabolite resulting in a loss of product yield.

There continues to be a need for new production and screening techniques that integrate production enhancement strategies into the primary screening techniques. The techniques are preferably non- destructive and rapid.

Brief Summary of the Invention

In one aspect, the present invention provides a process for producing a phytochemical. The process includes the step of culturing plant cells that produce the phytochemical in a multi-phase culture

medium that includes an aqueous phase and a hydrophobic phase. The hydrophobic phase has a high partition coefficient for the phytochemical relative to the aqueous phase such that the phytochemical produced by the cell culture is absorbed into the hydrophobic phase. In a preferred embodiment, the multi-phase culture medium is a two-phase culture medium. A preferred phytochemical is paclitaxel.

The hydrophobic phase preferably has a low partition coefficient for phytohormones and plant cell nutrients relative to the other phases. The hydrophobic phase preferably includes, high molecular weight alcohols, esters, ethers, triglycerides, halogenated solvents or silicones. An especially preferred hydrophobic phase includes 1 ,2,3,- trioctanoyl glycerol. In one embodiment, the hydrophobic phase is preconditioned with the aqueous phase.

The aqueous phase includes those factors necessary to sustain viability of the plant cell culture and optionally factors that stimulate phytochemical production. Such factors include phytohormones, the phytochemical biosynthetic precursors, elicitors and signal couplers.

Exemplary and preferred phytohormones include auxins, 2- napthyloxyacetic acid, 1-napthylacetic acid, cytokinins, kinetin, 6- benzylamino purine, 4-chlorophenoxyacetic acid, p- chlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, picloram, N ₆ -(2 -isopentyl)adenine, Zeatin and 6-(benzylamino)-9-(2- tetrahydropyranyl)-9H-purine. Exemplary and preferred biosynthetic precursors for paclitaxel include phenylalanine and gibberellic acid.

Exemplary and preferred elicitors for producing paclitaxel include extracts of Cytospora abietis or Penecillium minolυteum. An exemplary and preferred signal coupler for producing paclitaxel is jasmonic acid or a salt thereof.

A preferred plant cell culture for use in a process of the present invention to produce paclitaxel is a suspension culture of Taxus spp. cell lines. A paclitaxel production process of the present invention can further include the step of continuously extracting paclitaxel from the hydrophobic phase.

The present invention also provides for a non-destructive screening technique where production enhancement strategies are integrated into the screening technique. The screening technique is also non-destructive, and rapid. The cells produce the product in a multi-phase culture medium that includes an aqueous phase and a liquid hydrophobic phase. The hydrophobic phase has a high partition coefficient or higher affinity for the product than the aqueous phase such that product produced by the cell culture is extracted into the hydrophobic phase. In a preferred embodiment, the multi-phase culture medium is a two-phase culture medium. Preferred hydrophobic and aqueous phases are the same as set forth above.

Brief Description of the Drawings

In the drawings which form a portion of the specification:

FIG. 1 shows a schematic illustration of a two-phase culture medium for the production of paclitaxel.

FIG. 2 shows a schematic illustration of a two-phase culture medium for the production of paclitaxel with additional components for continuously extracting paclitaxel from the hydrophobic phase.

FIG. 3 shows an HPLC profile of paclitaxel in the medium (A) before extraction and (B) after extraction with 1 , 2, 3-trioctanoyl glycerol.

FIG. 4 shows an HPLC profile of second phase, 1 , 2, 3- trioctanoyl glycerol after in-situ extraction of Taxus suspension cell line. The cells had been growing in defined medium with the second phase present.

Detailed Description of the Invention

I. The Invention

The present invention relates to a novel process for producing phytochemicals of medicinal interest and the use of such a process in the screening of plant cell cultures to identify plant cell lines that produce a particular phytochemical of interest. An important aspect of the process is the use of a multi-phase culture medium that includes an aqueous phase and a hydrophobic phase. The phytochemical of interest is characterized by its partition into the hydrophobic phase.

In contrast to existing cell culture screening and production techniques, in which the cells producing phytochemical are destroyed, the present process is non-destructive as only the hydrophobic phase is analyzed for phytochemical content. Because the hydrophobic phase forms a distinct layer from the aqueous phase, sampling is easier and the desired phytochemical is separated from other medium constituents.

II. A Process of Producing Phytochemicals

In one aspect, therefore, the present invention provides a process for producing a desired phytochemical. The process includes the step of culturing plant cells that produce that phytochemical in a multi-phase culture medium that includes an aqueous phase and a hydrophobic phase. The hydrophobic phase has a high partition coefficient for the phytochemical relative to the aqueous phase such that the phytochemical produced by the cell culture is absorbed into the

hydrophobic phase. In a preferred embodiment, the multi-phase culture medium is a two-phase culture medium.

Any phytochemical that is partitionable between an aqueous and a hydrophobic phase can be produced by a process of the present invention. That phytochemical is produced and secreted by the particular plant cell culture. Phytochemicals produced and secreted by particular plant cell lines are well known in the art. A preferred phytochemical is paclitaxel, a drug used in cancer therapy.

Paclitaxel has been approved for the treatment of refractory ovarian and breast cancers and is in Phase I and Phase II triafs for the treatment of lung and colon cancers. At present, paclitaxel is derived from plant organs of Taxus species or by semi-synthetic conversion of paclitaxel intermediates isolated from natural sources. As an alternative to the current methods, paclitaxel may be produced by plant cell culture. Production of paclitaxel by cell culture provides the advantages of being more reliable, easier to validate, and is a renewable source as compared to isolation of the product from natural sources which lead to the destruction of the sources of the material. Paclitaxel is made by yew trees of the genus Taxus. Thus, a preferred plant cell culture for use in a process of the present invention to produce paclitaxel is a suspension culture of a Taxus spp. cell line. Exemplary such cell lines are from different organs of various species of yew such as Taxus brevifolia, Taxus media, Taxus baccata, Taxus cuspidata, and Taxus himalaya. Means for establishing plant cell cultures are well known in the art.

After selection of a cell line that produces and secretes a particular phytochemical, that cell line is cultured in a multi-phase culture medium that contains an aqueous phase for maintenance of the cells and a hydrophobic phase for partitioning of the phytochemical. As is well known in the art, the aqueous phase contains nutrients and

other factors needed to sustain plant cell viability. Those factors include carbon sources, salts and dissolved gases such as carbon dioxide. The use of Gamborg's B-5 medium supplemented with 30 g/L of sucrose is shown hereinafter in the Examples.

The aqueous phase optionally includes factors that stimulate the phytochemical production. Such factors include phytohormones, paclitaxel biosynthetic precursors of the phytochemical, elicitors and signal couplers.

Exemplary and preferred phytohormones include auxins, 2- napthyloxyacetic acid, 1 -napthylacetic acid, cytokinins, kinetin, 6- benzylamino purine, 4-chlorophenoxyacetic acid, p- chlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, picloram, N ₆ -(2 ₂ -isopentyl)adenine, Zeatin and 6-(benzylamino)-9-(2- tetrahydropyranyl)-9H-purine.

The auxins, 2-napthyloxyacetic acid (NOA) or 1 -napthylacetic acid (NAA) and the cytokinins, kinetin or 6-benzylamino purine (BAP) at a ratio of 10:1 molar ratio in the basic medium are effective in initiating cell lines that produce paclitaxel.

The induction of the synthesis of phytochemicals such as paclitaxel is often associated with the triggering of a plant's defense mechanism under attack from fungi, bacteria and viruses. Thus, stimulation of phytochemical synthesis can be accomplished by the addition to culture medium of certain elicitors, cell wall constituents of pathogens. Table 1 , below lists exemplary eiicitors that have been successfully used to enhance the biosynthesis of phytochemicals in plant cell cultures.

Table 1 Phytochemicals Elicited in Plant Cell Culture

Phytochemical Plant cell culture Elicitor source Enhancement (X-fold)

Harringtonine Cephaio Taxus Verticillium 51 alkaloids harringtinia dahliae

Medicarpin Cicer arietinυm S. cerevisiae 60

Diosgenin Diascorea Rhizopus deltoides arrhizus

Morphine Papaver Fusarium 20 somniferum moniliforme

Studies have shown that elicitors are likely useful in stimulating the phytochemical production with shorter production times than in non-elicited cultures. In particular, crude elicitor extracts prepared from the fungi Cytospora abietis and Penicillium minoluteum were able to stimulate paclitaxel production within 24 hours after addition (See U.S. Patent No. 5, 019,504).

Fungal cultures are grown in batch suspension cultures, culture filtrates harvested and mycelial mass homogenized. The culture filtrates and mycelial homogenate can be rotoevaporated to 1/20 of the original volume, then dialyzed to remove low molecular weight constituents. After centrifugation to remove insoluble material, the liquid is autoclaved and stored at -20°C prior to aseptic addition to cell cultures.

Means for determining optimal elicitor concentration, specificity, length of exposure, and timing of elicitor addition as a function of culture age, media composition, and cell line response are well known

in the art. Typical elicitor concentrations are 0.3-5% v/v of total culture volume. Typical exposure times are from about 24 to about 48 hours.

Biosynthetic precursors of phytochemicals enhance production. Exemplary such biosynthetic precursors for paclitaxel are phenylalanine and gibberellic acid. These precursors are typically added to the cell culture at the time the culture is initiated. Although paclitaxel production can occur in the absence of such biosynthetic precursors, there is typically an enhancement of production when precursors are utilized. Means for determining the optimal concentration of such precursors are well known in the art.

Gibberellic acid (GA ₃ ) has been shown to improve T. cuspidata cell growth rate without adversely affecting paclitaxel yield (Fett-neto, A.F., S.J. Melanson, K.Sakata, F. DiCosmo, Biotechnology, 11 , 731 , 1993). The effects of GA ₃ have been proposed to be due to synergistic effects with exogenous auxins and cytokinins in strongly promoting cell expansion.

Phenylalanine is the amino acid precursor for Winterstein acid, which is required for the biosynthesis of the sidechain of the paclitaxel molecule. It has been shown that the addition of 0.1 mM of phenylalanine to the medium significantly enhances paclitaxel yield in

T. cuspidata cultures without affecting the cell growth.

Jasmonic acid has lately been established as a signal coupler in the signal transduction process that takes place upon elicitation (See. e.g., Gundlach, H., M.J. Muller, T.M. Kutchan, M.H. Zenk, Proc. Natl. Acad. Sci. USA, 89, 2389, 1992 and Reinbothe, S., B. Mollenhauer, C.

Reinbothe, The Plant Cell, 6, 1197, 1994). Paclitaxel production can be enhanced upon elicitation of the Taxus cells.

Jasmonic acid has been proposed to be a second messenger in the elicitor induction of secondary metabolite biosynthesis. The effect of jasmonate on secondary metabolite gene expression has been noted in a wide cross-section of plants. Enhancement of secondary metabolites by addition of exogenous methylated jasmonate has been shown for a number of species (See, e.g., Gundlach, H., M.J. Muller, T.M. Kutchan, M.H. Zenk, Proc. Natl. Acad. Sci. USA, 89, 2389, 1992 and Reinbothe, S., B. Mollenhauer, C. Reinbothe, T e Plant Cell, 6, 1197, 1994).

The methyl ester of jasmonic acid, methyl jasmonate, is used for exogenous addition because the uptake of the methylated form by plant cells is thought to be more efficient than free jasmonic acid and it is readily available from commercial sources. The present inventors have shown that the addition of methylated jasmonate to cell cultures of Eschscholtzia californica enhances the yield of benzophenanthridine alkaloids (Dutta, A., Two-Phase Plant Cell Culture Integrated With Biosynthesis Enhancement Techniques, Ph.D. Thesis, Rutgers, The State University of New Jersey, New Brunswick, NJ, 1995).

There is usually synergism in the integration of in-situ extraction of secondary metabolites with such enhancement strategies as elicitation and precursor feeding. The biosynthesis of the alkaloids is increased substantially over constitutive levels by the addition of elicitor, precursors and methyl jasmonate. The magnitude of the feedback repression is greater than in control cultures or where elicitor and methyl jasmonate are added separately.

The culture medium includes a hydrophobic phase that absorbs the phytochemical produced and secreted by the plant cell culture into the aqueous medium. Absorption into the hydrophobic phase occurs because the hydrophobic phase has a higher partition coefficient for

the phytochemical than the aqueous phase. The removal of the phytochemical out of the aqueous phase and into the hydrophobic phase causes a shift in the equilibrium in the phytochemical synthesis towards more production and, thus, amplifies biosynthesis.

The components of the hydrophobic phase are non-toxic to the plant cells, and sufficiently selective for a particular phytochemical so as to partition the phytochemical into that phase. Hydrophobic phases that form an upper layer over the aqueous phase are preferable.

The hydrophobic phase preferably has a low partition coefficient for phytohormones, nutrients and other factors present in the aqueous phase. The absorption of such factors into the hydrophobic phase can be minimized by preconditioning the hydrophobic phase with the aqueous phase.

The hydrophobic phase preferably includes high molecular weight alcohols, hydrophobic esters, ethers, triglycerides, halogenated solvents, or silicones. An especially preferred hydrophobic phase for paclitaxel production includes 1 ,2,3-trioctanoyl glycerol. The use of 1 ,2,3-trioctanoyl glycerol to partition paclitaxel is described in detail hereinafter in the Examples. A schematic diagram of a two-phase culture medium, having an aqueous (A) and a hydrophobic (H) phase, for use in a process of the present invention is set forth in FIG. 1.

Means for determining a suitable hydrophobic phase are well known in the art. Briefly, an aqueous solution of the phytochemical is exposed to a hydrophobic phase and the distribution or partition of the phytochemical into the hydrophobic phase is determined. Samples of the hydrophobic phase are extracted with methanol and the insolubles removed. Extracts are then concentrated and assayed using well established HPLC methods, for example paclitaxel is easily identifiable

on this system. A photodiode array detector can be used to check the homogeneity of the paclitaxel peak and to provide a UV fingerprint to further verify paclitaxel's homogeneity. Final confirmation of the paclitaxel peak in HPLC can be carried out by LC-MS.

A production process of the present invention can further include the step of continuously extracting the phytochemical from the hydrophobic phase. An illustration of such a system for paclitaxel production is shown in FIG. 2

With reference to FIG. 2, a suitable plant cell culture is suspended in an aqueous phase (A). An upper layer is formed by a hydrophobic phase (H). Paclitaxel, secreted by the plant cells and partitioned into the hydrophobic phase, is routed (1) into a separator (e.g., a centrifugal separator) that separates paclitaxel and any aqueous media components that appear in the hydrophobic phase. The aqueous media components are recirculated (3) back into the aqueous phase of the culture reactor. Paclitaxel is routed (2) into an extractor (e.g., a hollow fiber) that removes the paclitaxel by methanol extraction. Components of the hydrophobic phase are then recirculated (4) back into the culture medium in the reactor. The extracted paclitaxel in methanol is then collected.

III. A Process of Screening Plant Cell Cultures to Identify Cultures that Produce Paclitaxel

A process of producing a phytochemical can be used to screen plant cell cultures to identify those that produce a particular phytochemical. In accordance with such a screening process, a plant cell culture suspected of producing a given phytochemical is cultured in a culture medium that includes an aqueous phase and a hydrophobic phase having a partition coefficient for that phytochemical higher than the aqueous phase

The culture is maintained for a period of time sufficient for phytochemical production and partition into the hydrophobic phase. The hydrophobic phase is then sampled and the presence of the phytochemical is determined in that sample, which presence indicates that the plant cell culture produces that phytochemical.

In a preferred embodiment, the phytochemical is paclitaxel and the cell lines are suspension cultures of Taxus spp. cell lines as set forth above. Preferred embodiments of aqueous and hydrophobic phase are the same as set forth above.

The following Examples illustrate preferred embodiments of the present invention and are not limiting of the specification and claims in any way.

EXAMPLE 1

The studies reported in this example show that paclitaxel can be removed from an aqueous medium by partitioning into a hydrophobic phase. The aqueous phase (medium) used in these studies was Gamborg's B-5 medium with 30 g/L of sucrose, vitamins and 25 μM of alpha-naphthalene acetic acid.

A stock solution of paclitaxel was prepared by dissolving 20 mg of crude paclitaxel in 10 mL of methanol. About 0.1 mL of this stock solution was dissolved in 150 mL of the aqueous medium. Analysis of the samples by HPLC showed that the paclitaxel content in the aqueous medium was 0.6 mg/L (FIG. 3).

Different aliquots of the paclitaxel-aqueous solution were adjusted to a pH value of 3.5, 5.45, 5.75, 6.36, or 6.9. 3 mL from each paclitaxel solution was mixed with 3 mL of a hydrophobic phase of 1 ,2,3-trioctanoyl glycerol (tricaprylin) and vortexed for 1 minute. The

phases were separated after 1 hour and the aqueous medium was analyzed by HPLC.

There was no detectable paclitaxel found in the aqueous medium after mixing with tπcaprylin indicating that tricaprylin completely removed paclitaxel from the aqueous medium.

EXAMPLE 2

Pieces of plant organs from Taxus species such as needles, leaves, stems, were placed on sterilized Gamborg's B-5 medium, with 30 g/L of sucrose, 0.8% w/w/ of agar, vitamins and 25 μM of alpha- naphthalene acetic acid and kept in an incubator at 22°C. Calli of undifferentiated cells grew on the plant organs after periods varying from two weeks to three months. T e calli were subcultured on the same medium.

Aliquots of the calli subculture were transferred to an aqueous culture medium (Gamborg's B-5 medium with 30 g/L of sucrose, vitamins and 25 μM of alpha-naphthalene acetic acid, pH 5.5) to establish suspension cultures. Those suspension cell cultures were then transferred to a two-phase culture medium containing Gamborg's B-5 medium as the aqueous phase and 10% v/v of tricaprylin as the hydrophobic phase.

The suspension cell cultures were grown in 125 mL shake flasks with 50 mL of culture medium in a gyratory shaker at 180 rpm at 25°C under conditions of darkness. Samples of the hydrophobic phase were analyzed for paclitaxel content by HPLC after 30 days of culture (FIG. 4).

The samples of the hydrophobic phase were extracted with methanol to remove any trapped aqueous phase prior to HPLC

analysis. The results showed that the hydrophobic phase contained paclitaxel in a concentration of 510 mg/L. Taken together with the data from Example 1 , these data show that plant cell cultures can be grown in a two-phase culture medium containing an aqueous and a hydrophobic phase and that paclitaxel produced and secreted by the plant cells is partitioned into the hydrophobic phase.