U.S. patent application number 13/618284 was filed with the patent office on 2013-01-17 for enhanced production of taxol and taxanes by cell cultures of taxus species.
This patent application is currently assigned to Phyton Holdings, LLC. The applicant listed for this patent is Venkataraman BRINGI, Prakash G. Kadkade, Christopher L. Prince, Braden L. Roach. Invention is credited to Venkataraman BRINGI, Prakash G. Kadkade, Christopher L. Prince, Braden L. Roach.
Application Number | 20130017582 13/618284 |
Document ID | / |
Family ID | 24619244 |
Filed Date | 2013-01-17 |
United States Patent
Application |
20130017582 |
Kind Code |
A1 |
BRINGI; Venkataraman ; et
al. |
January 17, 2013 |
ENHANCED PRODUCTION OF TAXOL AND TAXANES BY CELL CULTURES OF TAXUS
SPECIES
Abstract
This invention provides methods whereby taxol, baccatin III, and
other taxol-like compounds, or taxanes, can be produced in very
high yield from all known Taxus species, e.g., brevifolia,
canadensis, cuspidata, baccata, globosa, floridana, wallichiana,
media and chinensis. Particular modifications of culture conditions
(i.e., media composition and operating modes) have been discovered
to enhance the yield of various taxanes from cell culture of all
species of Taxus. Particularly preferred enhancement agents include
silver ion or complex, jasmonic acid (especially the methyl ester),
auxin-related growth regulators, and inhibitors of the
phenylpropanoid pathway, such as 3,4-methylenedioxy-6-nitrocinnamic
acid. These enhancement agents may be used alone or in combination
with one another or other yield-enhancing conditions. While the
yield of taxanes from plant cell culture of T. chinensis is
particularly enhanced by use of one or more of these conditions,
yield of taxanes for all Taxus species has been found to benefit
from use of these conditions.
Inventors: |
BRINGI; Venkataraman;
(Ithaca, NY) ; Kadkade; Prakash G.; (Marlboro,
MA) ; Prince; Christopher L.; (Lansing, NY) ;
Roach; Braden L.; (Interlaken, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BRINGI; Venkataraman
Kadkade; Prakash G.
Prince; Christopher L.
Roach; Braden L. |
Ithaca
Marlboro
Lansing
Interlaken |
NY
MA
NY
NY |
US
US
US
US |
|
|
Assignee: |
Phyton Holdings, LLC
San Antonio
TX
|
Family ID: |
24619244 |
Appl. No.: |
13/618284 |
Filed: |
September 14, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12972064 |
Dec 17, 2010 |
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13618284 |
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11836604 |
Aug 9, 2007 |
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12972064 |
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09083198 |
May 22, 1998 |
7264951 |
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11836604 |
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PCT/US97/08907 |
May 27, 1997 |
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09083198 |
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08653036 |
May 24, 1996 |
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PCT/US97/08907 |
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Current U.S.
Class: |
435/123 |
Current CPC
Class: |
C12P 17/02 20130101;
C12N 5/0025 20130101; C12P 15/00 20130101 |
Class at
Publication: |
435/123 |
International
Class: |
C12P 17/02 20060101
C12P017/02 |
Claims
1.-62. (canceled)
63. A method for producing paclitaxel and other taxanes from cell
culture of a Taxus species comprising: cultivating in suspension
culture, in a first medium developed for a growth phase and then in
a second medium developed for a product formation phase, cells of a
Taxus species derived from callus and/or suspension cultures, and
recovering said paclitaxel and other taxanes from said cells,
wherein said step of cultivating is carried out in the presence of
0.03% to 15% v/v of carbon dioxide in the gas phase in equilibrium
with at least one of said first medium and said second medium.
Description
[0001] This application is a continuation-in-part of International
application PCT/US97/08907, designating the U.S., filed May 27,
1997, and a continuation-in-part of U.S. Ser. No. 08/653,036, filed
May 24, 1996, which is a continuation-in-part of U.S. Ser. No.
08/370,494, filed Jan. 9, 1995, which is a divisional of U.S. Ser.
No. 07/874,344, now U.S. Pat. No. 5,407,816, filed Apr. 24, 1992,
which is a continuation-in-part of U.S. Ser. No. 07/839,144, filed
Feb. 20, 1992. The text of each priority application is expressly
incorporated herein by reference to the extent that the text of the
respective priority application differs from this application.
BACKGROUND OF THE INVENTION
[0002] A. Field of the Invention
[0003] This invention is directed to methods for the enhanced
production and recovery of taxol, baccatin III and other taxanes by
cell cultures of Taxus species.
[0004] B. Related Art
The Taxane Supply Challenge
[0005] Taxol is a diterpenoid alkaloid originally isolated from the
bark of the pacific yew, Taxus brevifolia (Wani, et al. 1971, J.
Am. Chem. Soc., 93, 2325-2327). Interest in taxol began when the
National Cancer Institute (NCI), in a large-scale screening
program, found that crude bark extracts exhibited anti-tumor
activities. Since then, clinical trials have confirmed that taxol
is extremely effective against refractory ovarian cancers, and
against breast and other cancers. Taxol has been pronounced as a
breakthrough in chemotherapy because of its fundamentally different
mechanism of cytotoxicity, i.e., by inhibiting depolymerization of
microtubules (see Rowinsky, et al., 1990, J. Natl. Cancer Inst.,
82, 1247-1259).
[0006] A daunting variable in the taxol equation has been supply.
Bark-derived taxol has been discontinued as the primary source of
commercial drug; large-scale production has been achieved by
semi-synthesis, i.e., chemical attachment of a side chain to the
plant-derived precursor, 10-deacetylbaccatin III. Total synthesis,
while accomplished by academic laboratories, shows little promise
as a viable commercial route to taxol. There is therefore an urgent
need to develop cost-effective, environmentally-benign, and
consistent sources of supply to keep up with the growing demand for
taxol.
[0007] In addition to taxol, there is an urgent need to develop
processes for the commercial production of related taxane
molecules. Derivatives of taxol such as Taxotere have already been
introduced into the world market. Further, tremendous research
activity is being focused on the discovery and development of novel
taxane derivatives with advantageous activity. These advances are
likely to create an ongoing need for large quantities of an
appropriate starting "skeleton" molecule from which any given
derivative could be effectively synthesized.
[0008] One example of such a molecule is the aforementioned
precursor, 10-deacetylbaccatin III, which is used as the starting
point for semi-synthetic taxol. Another desirable starting molecule
for semi-synthetic production of taxol and other derivatives is
baccatin III. Baccatin III is normally not accumulated as a major
taxane in planta, and hence there is no facile large-scale natural
source for this molecule. However, it is a very desirable starting
point for semi-synthesis because of its chemical closeness to
taxol; for example, the steps that are required for acetylation of
the 10 position of 10-deacetylbaccatin III are circumvented if
baccatin III is the starting point rather than 10-deacetylbaccatin
III.
[0009] This invention is related to the development of plant cell
culture-based processes for the commercial production of taxol,
baccatin III and other taxanes.
Tissue Cultures as a Source of Plant-Derived Chemicals
[0010] The ability of plant cells to divide, grow, and produce
secondary metabolites under a variety of different cultural regimes
has been amply demonstrated by a number of groups. At present, two
compounds, shikonin (a red dye and anti-inflammatory) and
ginsengoside (a tonic in oriental medicine) are produced by
tissue-culture processes in Japan. Many other processes are
reportedly close to commercialization, including vanillin,
berberine and rosmarinic acid (see Payne, et al. 1991, "Plant Cell
and Tissue Culture in Liquid Systems," Hanser Publishers,
Munich).
[0011] The advantages of a plant cell culture process for taxol,
baccatin III, and taxanes are many: (i) A cell culture process
ensures a limitless, continuous and uniform supply of product, and
is not subject to pests, disasters and seasonal fluctuations, (ii)
cell cultures can be cultivated in large bioreactors, and can be
induced to overproduce the compound of interest by manipulating
environmental conditions, (iii) cell cultures produce a simpler
spectrum of compounds compared to bark or needles, considerably
simplifying separation and purification, (iv) a cell culture
process can adapt quickly to rapid changes in demand better than
agriculture-based processes, (v) besides supplying taxol, baccatin
III or other precursors, a cell culture process could also produce
taxane compounds that exhibit advantageous bioactivity profiles, or
that could be converted into other bioactive derivatives.
[0012] Since aseptic, large-scale, plant cell cultivation is
inherently expensive, a cell culture process becomes commercially
relevant only when these costs are offset by high productivity.
Every plant species and target metabolite is different, and
different approaches are necessary for every particular system.
This invention focuses on creative and skilled approaches for
obtaining highly productive plant cell cultures for taxol, baccatin
III, and taxane production.
Problems with Tissue Cultures of Woody Plants and Conifers
[0013] A historical survey of the literature suggests that whereas
herbaceous plants have been relatively easily manipulated in
culture, productive cultures of woody plants and conifers have been
achieved only with difficulty.
[0014] The growth of secondary metabolite producing gymnosperm- and
conifer-cultures have been generally low. For example, Berlin and
Witte, (1988, Phytochemistry, 27, 127-132) found that cultures of
Thuja occidentalis increased their biomass by only ca. 30% in 18
days. Van Uden et al. (1990, Plant Cell Reports, 9, 257-260)
reported a biomass increase of 20-50% in 21 days for suspensions of
Callitris drummondii. Westgate et al. (1991, Appl. Microbiol.
Biotechnol., 34, 798-803) reported a doubling time of ca. 10 days
for suspensions of the gymnosperm, Cephalotaxus harringtonia. As
summarized by Bornman (1983, Physiol. Plant. 57, 5-16), a
tremendous amount of effort has been directed towards medium
development for spruce suspensions (Picea abies). This collective
work demonstrates that gymnosperm suspensions are indeed capable of
rapid growth, but that no generalities can be applied, and that
media formulations for different cell lines must be optimized
independently.
[0015] A survey of secondary metabolite productivity among
gymnosperm cultures also points to the difficulty of inducing rapid
biosynthesis compared to herbaceous species. For example, cultures
of Cephalotaxus harringtonia produced terpene alkaloids at a level
of only 1% to 3% of that found in the parent plant (Delfel and
Rothfus, 1977, Phytochemistry, 16, 1595-1598). Even upon successful
elicitation, Heinstein (1985, Journal of Natural Products, 48, 1-9)
was only able to approach the levels produced in the parent plant
(ca. 0.04% dry weight total alkaloids). Van Uden et al (1990) were
able to induce suspension cultures of the conifer Callitris
drummondii to produce podophyllotoxin, but only at levels one tenth
of that produced by the needles. The ability of Thuja occidentalis
to produce significant levels of monoterpenes (10-20 mg/L) and the
diterpenoid dehydroferruginol (2-8 mg/L) has been convincingly
demonstrated by Berlin et al. (1988). However, these results were
obtained with a slow-growing (30% biomass increase in 18 days) and
low cell density (5 to 7 grams dry weight per liter) culture.
Cell Culture for Taxane Production
[0016] The difficulties in achieving rapid growth and high
productivity encountered in gymnosperm-suspensions have generally
been reflected in the reports so far on taxane production in Taxus
cell cultures.
[0017] Jaziri et al. (1991, J Pharm. Belg., 46, 93-99) recently
initiated callus cultures of Taxus baccata, but were unable to
detect any taxol using their immunosorbent assay. Wickremesinhe and
Arteca (1991, Plant Physiol., 96, (Supplement) p. 97) reported the
presence of 0.009% thy weight taxol in callus cultures of Taxus
media (cv. hicksii), but details on the doubling times, cell
densities, and the time-scale over which the reported taxol was
produced, were not indicated.
[0018] U.S. Pat. No. 5,019,504 (Christen et al. 1991) describes the
production and recovery of taxane and taxane-like compounds by cell
cultures of Taxus brevifolia. These workers reported taxol
production at a level of 1 to 3 mg/L in a two- to four-week time
frame. They also reported a cell mass increase of "5-10 times in
3-4 weeks", which corresponds to doubling times of ca. 7 to 12
days.
[0019] Significant increases in taxane titers and volumetric
productivity are required before an economically-viable plant cell
culture process for taxane production can supply the projected
annual demand of many hundreds of kilograms per year.
SUMMARY OF THE INVENTION
[0020] The objects of this invention include the formulation of
special environmental conditions to foster rapid growth, high cell
densities, and high cell viabilities. (The growth characteristics
reported in this study surpass previous results by a significant
factor.)
[0021] An object of this invention is to produce taxanes at high
rates by careful selection of cell lines, careful choice and
manipulation of medium conditions, incorporation of enhancement
agents, and careful selection of process-operating modes.
[0022] The objects of this invention include the ability to
manipulate the profile of taxanes produced by altering media
formulations and environmental conditions. In particular, it is an
object to encourage cells to produce taxol or baccatin III as the
predominant taxane product, and/or to suppress the production of
the by-product cephalomannine, thereby providing an elegant
biological solution to an expensive and important downstream
separation and purification problem. These and other objects are
met by one or more of the embodiments of this invention.
[0023] The inventors have discovered that taxol, baccatin III, and
other taxol-like compounds, or taxanes, can be produced in very
high yield from all known Taxus species, e.g., brevifolia,
canadensis, cuspidata, baccata, globosa, floridana, wallichiana,
media and chinensis. Further, by the methods of this invention it
is possible to obtain taxol, baccatin III, and other taxanes in a
much shorter time frame than previously reported. In particular,
the inventors found that the species, Taxus chinensis, is capable
of rapid growth and of producing extremely high levels of taxol,
baccatin III, and taxanes within a short period of time. With the
species Taxus chinensis, the inventors have been able to manipulate
cells to yield taxol, baccatin III, and taxanes in amounts far in
excess of the amounts obtained from tissue cultures of the other
Taxus species.
[0024] Particular modifications of culture conditions (i.e., media
composition and operating modes) have been discovered to enhance
the yield of various taxanes from cell culture of all species of
Taxus. Particularly preferred enhancement agents include silver ion
or complex, jasmonic acid (especially the methyl ester),
auxin-related growth regulators, and inhibitors of the
phenylpropanoid pathway, such as 3,4-methylenedioxy-6-nitrocinnamic
acid. These enhancement agents may be used alone or in combination
with one another or other yield-enhancing conditions. While the
yield of taxanes from plant cell culture of T. chinensis is
particularly enhanced by use of one or more of these conditions,
yield of taxanes for all Taxus species has been found to benefit
from use of these conditions.
[0025] In one embodiment, this invention provides a method for
producing taxanes in high yields in cell culture of a Taxus species
comprising cultivating cells of a Taxus species in suspension
culture in one or more nutrient media under growth and product
formation conditions, and recovering one or more taxanes from said
cells or said medium of said cell culture, or both, the cells being
derived from callus or suspension cultures and the nutrient media
containing an inhibitor of phenylpropanoid metabolism. Suitable
inhibitors of phenylpropanoid metabolism include
3,4-methylenedioxy-6-nitrocinnamic acid, 3,4-methylenedioxycinnamic
acid, 3,4-methylenedioxy-phenylpropionic acid,
3,4-methylenedioxyphenylacetic acid, 3,4-methylenedioxybenzoic
acid, 3,4-trans-dimethoxycinnamic acid, 4-hydroxycinnamic acid,
phenylpropiolic acid, fluorophenylalanine, 1-aminobenzotriazole,
2-hydroxy-4,6-dimethoxybenzoic acid, SKF-525A, ammonium oxalate,
vinylimidazole, diethyldithiocarbamic acid, and sinapic acid.
[0026] In a preferred embodiment, at least one of the one or more
nutrient media used in the method of this invention also comprises
another enhancement agent which may be an inhibitor of ethylene
action; jasmonic acid or an ester of jasmonic acid; or an
auxin-related growth regulator. In particularly preferred
embodiments, the other enhancement agent is an inhibitor of
ethylene action which is a silver-containing compound, or a silver
complex, or a silver ion. In another particularly preferred
embodiment, the other enhancement agent is jasmonic acid or an
alkyl ester thereof, and more preferably, the alkyl group
esterified to jasmonic acid has from one to six carbon atoms. In an
even more preferred embodiment, the enhancement agent is jasmonic
acid or an alkyl ester thereof, and the medium also contains a
silver-containing compound, a silver complex or silver ion. In yet
another particularly preferred embodiment, the other enhancement
agent is an auxin-related growth regulator, such as indoleacetic
acid, picloram, .alpha.-naphthaleneacetic acid, indolebutyric acid,
2,4-dichlorophenoxyacetic acid, 3,7-dichloro-8-quinolinecarboxylic
acid, or 3,6-dichloro-o-anisic acid.
[0027] In another embodiment, this invention provides a method for
producing taxanes in high yields in cell culture of a Taxus species
by cultivating cells of a Taxus species in suspension culture in
one or more nutrient media under growth and product formation
conditions, and recovering one or more taxanes from said cells or
said medium of said cell culture, or both, the cells being derived
from callus or suspension cultures and the nutrient media
containing silver at a concentration of 900 .mu.M or less in the
form of a silver-containing compound, or a silver complex, or a
silver ion, along with at least one enhancement agent which may be
jasmonic acid or an ester of jasmonic acid an auxin-related growth
regulator. In a preferred embodiment, the enhancement agent is
jasmonic acid or an ester of jasmonic acid, and the molar ratio of
silver to enhancement agent is less than 9.5. In another preferred
embodiment, the enhancement agent is an auxin-related growth
regulator, and the molar ratio of silver to enhancement agent is at
lease 0.011.
[0028] In any of the above embodiments, the one or more nutrient
media may also include a taxane precursor, which may be
.alpha.-phenylalanine, .beta.-phenylalanine, or a mixture thereof.
In any of the above embodiments, the one or more nutrient media may
also include glutamine, glutamic acid, aspartic acid or a mixture
of these amino acids, or one or more nutrient media used in
cultivation of the cells may include maltose, sucrose, glucose
and/or fructose as a carbon source, preferably as the primary
carbon source. In one embodiment, the nutrient medium is the same
for cell culture growth and for taxol and taxane production. In an
alternative embodiment, production of one or more taxanes is
induced in the culture by changing the composition of the nutrient
medium. In a preferred embodiment, the medium in the culture is
periodically exchanged, and typically the medium exchange
accomplishes periodic removal of taxanes from the culture.
Preferably, cells of said Taxus species are cultivated by a
fed-batch process.
[0029] Typically, taxol or baccatin III and/or other taxanes are
recovered from said cells or said medium of said cell culture, or
both. Generally, cultivation of Taxus species according to this
invention provides an average volumetric productivity of taxanes
which is at least 15 mg/L/day averaged over the period of taxane
production. The average volumetric productivity of taxol is
typically at least 10 mg/L/day computed for the period of taxol
production. The average volumetric productivity of baccatin III is
typically at least 15 mg/L/day computed for the period of taxane
production.
[0030] Preferably, cells cultured according to the method of this
invention are cells of Taxus species, and the species may be T.
brevifolia, T. canadensis, T. chinensis, T. cuspidata, T. baccata,
T. globosa, T. floridana, T. wallichiana, or T. media. Preferably,
the cells of a Taxus species used in the method of this invention
are cells which produce taxol above background by ELISA in callus
culture or suspension culture in medium that contains no
enhancement agents. More preferably, the cells of a Taxus species
used in the method of this invention are cells which produce
taxanes in suspension culture at an average volumetric productivity
of 10 mg/L in a medium containing silver thiosulfate, methyl
jasmonate and auxin.
DESCRIPTION OF THE FIGURES
[0031] FIG. 1. Biomass increase in a Taxus chinensis suspension
culture line K-1 over a typical batch growth cycle in Medium A.
Error bars represent the standard deviation measured from duplicate
flasks.
[0032] FIG. 2. Effect of medium exchange on days 9 and 12 on taxol
(A) and total taxane (B) productivity in a 15-day experiment. The
numbers in each box represent the time interval (days) over which
the product was produced. The darkened portion of the intracellular
boxes represents the taxol or total taxanes that were present in
the cell inoculum at the start of the experiment. All treatments
were performed in duplicate. Taxus chinensis suspension cell line
K-1 was used with Medium A as elaborated in Table 2.
[0033] FIG. 3. Spectral characteristics of a Standard Gro-Lux lamp
(GTE Sylvania, Danvers, Mass.) used in Example 7.3.
[0034] FIG. 4. Taxane production in Taxus chinensis cell suspension
K-1. The portion of the chromatogram from 10 to 40 minutes is
shown. Diode array scans of selected taxane peaks show a
characteristic taxane UV absorption spectrum, with a peak at 227
nm.
[0035] FIG. 5. Taxol and taxane production after prolonged
cultivation in Medium C by Taxus chinensis cell line K-1. The upper
panel tabulates the data for the known and unknown taxanes, whereas
the lower panel shows incremental taxol and taxane production in
the 25 to 42 day time period.
[0036] FIG. 6. MS/MS confirmation of taxol in cell culture
supernatant. Panel A shows the ion spray APCI mass spectrum of
authentic taxol and panel B shows the daughter ion spectrum of the
parent peak (m/z 871=taxol+NH4+). Panel C represents the ion spray
APCI spectrum from a crude cell culture extract and shows m/z 854
and 871 characteristic of taxol. Panel D shows the corresponding
daughter spectrum of m/z 871 and provides unequivocal evidence for
the presence of taxol in cell culture supernatant.
DETAILED DESCRIPTION OF THE INVENTION
[0037] Plants have long provided important sources of
pharmaceuticals and specialty chemicals. These products have
typically been obtained through extraction of the harvested plant
materials or by chemical synthesis. Taxol and taxanes have become
one of the most important class of anticancer agents to recently
emerge from the screening of natural products.
[0038] As used herein, the terms taxol-like compounds, or taxanes,
are used interchangeably to describe a diterpenoid compound with a
taxane ring. The taxanes may themselves possess antineoplastic
activity, or may be modified to yield bioactive compounds. The term
total taxanes refers to all taxanes that exhibit a characteristic
UV absorbance as described in Example 5 below.
[0039] As used herein, the term "callus" is used to describe a mass
of cultured plant cells that is structurally undifferentiated, and
is cultivated on solidified medium. As used herein, the term
"suspension culture" is used to describe structurally
undifferentiated cells that are dispersed in a liquid nutrient
medium. It is understood that suspension cultures comprise cells in
various stages of aggregation. A range of aggregate sizes are
encountered in the suspensions described in this invention, with
sizes ranging from tens of microns in diameter (single cells or
few-aggregated cells) to aggregates many millimeters in diameter,
consisting of many thousands of cells.
[0040] The plant material useful in this invention may be obtained
from any known Taxus species, e.g., brevifolia, canadensis,
cuspidata, baccata, globosa, floridana, wallichiana (also referred
to as yunnanensis), media, fastigiata and chinensis (including the
synonymous species, such as sumatrama, celebica, and speciosa, and
the subspecies chinensis var. mairei). In particular, the inventors
have identified the species Taxus chinensis as capable of producing
significant quantities of taxol, baccatin III, and taxanes at high
volumetric productivities.
[0041] It has been found by the inventors that specific taxane
content varies with plant species, and within plant species from
tissue source and specific trees. Selecting a high yielding source
and culture for taxane production is an important first step
towards providing sufficient quantities of taxanes for therapeutic
use.
Benchmarks for Commercial Relevance
[0042] A number of benchmarks may be used to gauge the commercial
attractiveness and viability of a given plant-cell-culture-based
process for taxane production. The benchmarks should characterize
and underpin the key performance parameters of the process,
including fermentation costs, the ease of downstream recovery, and
the capacity of production. The benchmarks that will be described
here are the broth titer and the volumetric productivity.
[0043] The broth titer is defined as the concentration of product
in the whole broth, and is usually expressed as milligrams of
product per liter of broth (mg/L). By definition, the whole broth
titer does not distinguish between the intracellular and
extracellular portions of the product. The broth titer is typically
used to characterize the performance of a batch or fed-batch
process. A higher broth titer implies a greater production capacity
for a given reactor volume, and concomitantly, lower unit
production costs. Similarly, a high-titer product is usually easier
to recover in high yield, thus leading to further improvements in
unit production costs.
[0044] The volumetric productivity is defined as the amount of
product produced per unit reaction volume per unit time, and is
commonly expressed in units of milligrams per liter per day. For
the purposes of taxane production, the time scale is defined as the
time frame during which production takes place at the production
scale immediately preceding harvest and recovery. The volumetric
productivity complements the titer as a benchmark for batch and
fed-batch processes, and is particularly useful for characterizing
processes where the product is removed during production, for
example, by periodic medium exchange or another method of removal.
A high volumetric productivity implies greater production capacity
for a given reactor volume over a give time period, and
concomitantly, lower unit production costs and greater overall
process performance.
[0045] In certain cases the volumetric productivity is used to
gauge the intrinsic capability of a biological process--for
example, in the earlier stages of process development, it is useful
to measure the productivity over the most productive part of the
production cycle, i.e., over a short time period when the rates of
biosynthesis are at their highest. This is typically referred to as
the maximal instantaneous volumetric productivity. However, in
gauging the performance of a process, the more appropriate
benchmark is the average volumetric productivity in which the
productivity is measured over the entire productive phase. Clearly,
in order to achieve the highest average volumetric productivity,
the maximal instantaneous productivity must be maintained through
the majority of the productive phase. Unless otherwise qualified,
the term volumetric productivity refers to the average volumetric
productivity, determined for the entire production phase Typically,
production phase is initiated by changes in nutrient medium
composition, either by replacing growth medium with production
medium or by adding enhancement agents which induce a significant
enhancement in taxane production.
Initiation of Taxus Cell Lines
[0046] Taxus plant material may be collected from all over North
America as well as from other continents. The culture is initiated
by selecting appropriate Taxus tissue for growth. Tissue from any
part of the plant, including the bark, cambium, needles, stems,
seeds, cones, and roots, may be selected for inducing callus.
However, for optimum yield of taxol, needles and meristematic
regions of plant parts are preferred. Most preferred are new growth
needles (e.g., one to three months old), which can generally be
identified by a lighter green color. The term "new growth" is
broadly intended to mean plant needle production within that year's
growing season.
[0047] To prevent contamination of the culture, the tissue should
be surface-sterilized prior to introducing it to the culture
medium. Any conventional sterilization technique, such as CLOROX (a
trademark owned by the Clorox Company for bleach) treatment would
be effective. In addition, antimicrobial agents such as cefoxitin,
benlate, cloxacillin, ampicillin, gentamycin sulfate, and
phosphomycin may be used for surface sterilization of plant
material.
Callus Growth
[0048] Cultures will typically exhibit variability in growth
morphology, productivity, product profiles, and other
characteristics. Since individual cell lines vary in their
preferences for growth medium constituents, many different growth
media may be used for induction and proliferation of the
callus.
[0049] The appropriate medium composition varies with the species
being cultured. The preferred media for the different species are
listed in Table 3. For example, although others may be used, the
preferred growth nutrient media for Taxus chinensis are A, D, I, J,
K, L, M, O, P. These media preferably contain the ingredients
listed in Table 2. Cultures are preferably carried out with medium
components incorporated at the levels shown in Table 2, although
the skilled artisan will recognize that some variation in these
levels will not adversely affect cell growth. For example, when
medium A is used, growth hormones or regulators incorporated into
the medium in an amount between 1 ppb to 10 ppm, and preferably at
2 ppb to 1 ppm. When medium D is used, the growth hormones or
regulators are incorporated at levels ranging from 1 ppb to 10 ppm,
and preferably at 2 ppb to 2 ppm. The amounts of other medium
ingredients can be incorporated at levels ranging from 1/10th
concentration to three times the concentrations indicated in Table
2.
[0050] Production of taxanes in large quantities is facilitated by
cultivating Taxus cells in suspension culture. Generally,
suspension culture can be initiated using a culture medium that was
successful in callus culture. However, the requirements for
suspension culture, and particularly for highly efficient
production of taxanes, may be better met by modification of the
medium. It has been found that when Taxus cells are cultured in
modified culture medium and processing parameters tailored
according to the method of this invention, the yield of one or more
taxanes from the culture is substantially increased.
[0051] As used herein, the term "nutrient medium" is used to
describe a medium that is suitable for the cultivation of plant
cell callus and suspension cultures. The term "nutrient medium" is
general and encompasses both "growth medium" and "production
medium". The term "growth medium" is used to describe an nutrient
medium that favors rapid growth of cultured cells. The term
"production medium" refers to an nutrient medium that favors taxol,
baccatin III, and taxane biosynthesis in cultured cells. It is
understood that growth can occur in a production medium, and that
production can take place in a growth medium; and that both optimum
growth and production can take place in a single nutrient
medium.
Suspension Growth
[0052] Taxus suspension cultures are capable of rapid growth rates
and high cell densities like other plant cell cultures. However,
optimal conditions may vary from one cell line to another, and
accordingly, methods leading towards rapid optimization for any
given cell line must be considered.
[0053] The cultures of various Taxus species are cultivated by
transfer into nutrient media containing macro- and micro-nutrient
salts, carbon sources, nitrogen sources, vitamins, organic acids,
and natural and synthetic plant growth regulators. In particular,
nutrient medium for suspension culture of Taxus cells will
typically contain inorganic salts that supply the macronutrients
calcium, magnesium, sodium, potassium, phosphate, sulfate,
chloride, nitrate, and ammonium, and the micronutrients such as
copper, iron, manganese, molybdenum, zinc, boron, cobalt, iodine,
and nickel. The medium will also typically contain vitamins such as
myo-inositol, thiamine, ascorbic acid, nicotinic acid, folic acid,
pyridoxine and optionally biotin, pantothenate, niacin and the
like. These components may be present at concentration ranges of
1/30th to thirty times the concentrations listed in Table 2, and
preferably at 1/20th to twenty times the concentrations listed in
Table 2, more preferably at 1/3 to three times the concentrations
listed in Table 2, and most preferably at the concentrations listed
in Table 2.
[0054] The nutrient medium will also contain one or more carbon
sources, and will typically contain a primary carbon source, which
is defined as a source that provides over 50% of the total carbon
in the nutrient medium. The primary carbon source is preferably
lactose, galactose, raffinose, mannose, cellobiose, arabinose,
xylose, sorbitol, or preferably glucose, fructose, sucrose or
maltose. The concentration of the primary carbon source may range
from 0.05% (w/v) to 10% (w/v), and preferably from 0.1% (w/v) to 8%
(w/v).
[0055] The nutrient medium will also contain a nitrogen source,
which, in addition to any nitrogen added in the form of
macronutrient salts, will preferably be provided at least in part
by an organic nitrogen source (e.g., one or more amino acids such
as glutamine, glutamic acid, and aspartic acid, or protein
hydrolyzates). These organic nitrogen sources may supply nitrogen
at concentrations ranging from 0.1 mM to 60 mM, and preferably from
1 to 30 mM. The medium may also contain one or more organic acids
such as acetate, pyruvate, citrate, oxoglutarate, succinate,
fumarate, malate, and the like. These components may be included in
the medium at concentrations of 0.1 mM to 30 mM, and preferably at
concentrations of 0.5 mM to 20 mM.
[0056] The medium will also typically contain one or more natural
or synthetic plant growth regulators, including auxin-related
growth regulators such as picloram, indoleacetic acid,
1-naphthaleneacetic acid, indolebutyric acid,
2,4-dichlorophenoxyacetic acid, 3,7-dichloro-8-quinolinecarboxylic
acid, 3,6-dichloro-o-anisic acid, and the like, cytokinin-related
growth regulators such as N.sup.6-benzyladenine,
6-[.gamma.,.gamma.-dimethylallylamino]purine, kinetin, zeatin,
N-phenyl-N'-1,2,3-thidiazol-5-ylurea (thidiazuron) and related
phenylurea derivatives and the like, gibberrellins such as
GA.sub.3, GA.sub.4, GA.sub.7, and GA derivatives, abscisic acid and
its derivatives, brassinosteroids, and ethylene-related growth
regulators. Additional suitable auxin-related plant growth
regulators are listed below. It should be noted that the nutrient
medium may contain more than one growth regulator belonging to a
single class, for example, more than a single auxin-related
regulator, or more than one cytokinin-related regulator. The growth
regulators will be preferably incorporated into the medium at a
concentration between 10.sup.-10 M to 10.sup.-3 M, preferably at
10.sup.-8 to 3.times.10.sup.-5 M, and more preferably at the
concentrations listed in Table 2.
[0057] Unless otherwise indicated, growth media as defined herein
provide a suitable starting point for routine optimization of
callus culture media and production media. It is a routine matter
for those skilled in the art to incorporate, modify, and manipulate
particular classes of components, and components from within a
given class, to achieve optimum performance; particular media
modifications are provided in the Tables and Examples below.
[0058] The liquid cultures are exposed to a gascous environment
such as air and preferably shaken or otherwise agitated to allow
for proper mixing of culture components. The cultures are
maintained at a temperature between 23.degree. C. and 27.degree.
C., although under appropriate conditions and/or circumstances,
temperatures could range from 0.degree. C. to 33.degree. C. The pH
may be from about 3 to 7 and preferably between 4 to 6. The culture
may be grown under light conditions ranging from total darkness to
total light (narrow band and/or broad spectrum) for various periods
of time.
[0059] Doubling times have been measured by monitoring
time-dependent biomass increase, as well as by simply monitoring
the growth index during routine subculture. Maximum dry weight
densities of 15-24 grams per liter have been achieved. The growth
characteristics of various Taxus species suspensions are elaborated
in Example 4.
Taxane Production Conditions
[0060] If secondary metabolite formation in a suspension culture
takes place concurrently with growth, the metabolite is termed
growth-associated, and a single medium formulation may be
sufficient to achieve good growth and high level production. In
many other systems, it has been found that rapid growth and high
product formation do not take place concurrently. In such cases,
growth and production phases are separated and a medium for each
phase is developed independently (reviewed in Payne et al. 1991,
Plant Cell and Tissue Culture in Liquid Systems, Hanser publishers,
Munich). In the case of taxane production in Taxus, growth and
product formation can be separated, and independent media have been
developed for each.
[0061] In a preferred mode of this invention, the composition of
the medium during the cell growth phase is different from the
composition of the medium during the taxane production phase. For
example, the identity and level of the carbon sources, particularly
the primary carbon source, may change between the growth phase and
the production phase. Preferably the production medium will contain
sugar at a level higher than that of the growth medium. More
preferably the initial sugar level in the production medium may be
2-20 times higher in the production phase than the growth phase.
The primary carbon source is preferably lactose, galactose,
raffinose, mannose, cellobiose, arabinose, xylose, sorbitol, or
preferably glucose, fructose, sucrose or maltose. The concentration
of the primary carbon source may range from 0.05% (w/v) to 10%
(w/v), and preferably from 0.1% (w/v) to 8% (w/v). Particularly
preferred carbon sources for production of taxol or baccatin are
maltose, sucrose, glucose and/or fructose. In particularly
preferred embodiments, these sugars will be incorporated in initial
nutrient medium at concentrations of at least 3.5%.
[0062] The identity and the level of organic supplements, which may
include, vitamins, organic nitrogen sources such as amino acids, as
well as the presence or levels of the enhancement agents described
below, may change or may differ in the media. The identity and
levels of the natural or synthetic plant growth regulators may
differ between the media. Similarly the levels and identity of
macronutrient and micronutrient salts may also differ between the
growth and production media. Preferably, the salt content is
reduced in the production medium relative to the growth medium,
optionally, nitrate and sulfate salts are reduced
disproportionately and more preferably the extent of reduction is a
reduction by a factor of 2-20 fold. However, it is understood that
a single growth/production medium may be formulated for this
culture.
[0063] The production media developed here not only increase taxane
formation, but also direct cellular biosynthesis towards production
of particular taxanes, such as taxol or baccatin III. In addition,
production of interfering by-products such as cephalomannine is
minimal compared to bark tissue. The production media developed
here also promote prolonged cell viability and biosynthesis, and in
addition, cause significant levels of product to be secreted into
the extracellular medium. These characteristics are extremely
important in the operation of an efficient commercial scale process
for taxane production.
[0064] Methods for the extraction and recovery of taxol and taxanes
from cells and the medium follow conventional techniques (see,
e.g., Example 5). The immuno-assay (ELISA) technique largely
followed the protocols supplied by Hawaii Biotechnology in the
commercially available kit (see also, Grothaus et al. 1995, Journal
of Natural Products, 58, 1003-1014 incorporated herein by
reference). The antibody may be specific for any taxane, such as
taxol or baccatin III, or less specifically, for the taxane
skeleton. High performance liquid chromatography methods were
slightly modified from existing protocols as elaborated in Example
5. Under the conditions used in this invention, clear resolution of
taxane peaks was achieved, resulting in accurate detection and
quantitation. Because of the possibility of co-eluting non-taxane
components, the spectral purity of taxane peaks were routine by
checked by diode array before integration of peak areas. Retention
times of taxane standards are listed in Example 5, and a sample
chromatogram is included in FIG. 4.
[0065] For higher plants, light is a potent factor in secondary
metabolism both in intact plant as well as in cell cultures. Both
the intensity and wavelength of light are important (Seibert and
Kadkade 1980, "Plant Tissue Culture as a Source of Biochemicals."
E. J. Staba (ed), CRC Press, Boca Raton, Fla., pp. 123-141). For
example, flavanoid and anthocyanin biosynthesis are usually favored
by high intensity continuous light, while dark-cultivated cultures
may be preferable for other metabolites. Increase in greening or
photosynthetic capacity of cultured cells may also increase product
formation or product spectrum. The inventors' studies involved the
use of broad-band and well as specific narrow-band light sources.
As shown in Example 7.3, light exposure can bring about increased
taxol accumulation as well as secretion into the medium. The
stimulatory effect of light on taxol production suggests the
existence of unique control mechanisms for biosynthesis of taxanes.
The nature of the photoreceptor and biochemical characteristics of
light-induced stimulation are not yet clear. However, the
incorporation of enhancement agents, in accordance with the
teachings of this invention, render the role of light as less
critical for optimum performance.
[0066] In addition to non-volatile dissolved nutrients, gaseous
components, primarily oxygen, carbon dioxide, and ethylene (a plant
hormone), play critical roles in growth and product formation. Two
parameters are important. The dissolved gas concentrations favoring
growth and taxol formation are obviously important since they
dictate reactor operating conditions. In addition, the rates of
consumption or production need to be incorporated into reactor
design, so that the optimum specified concentrations can be
maintained.
[0067] Besides its importance in respiration, oxygen can also
dramatically affect the rate of secondary metabolite biosynthesis.
A high saturation constant for an oxygen-requiring step on a
secondary biosynthetic pathway may require cells to be subjected to
high oxygen levels in the reactor. The importance of CO.sub.2
supplementation in maintaining high growth rates has been
documented. Ethylene, a plant hormone, plays pleiotropic roles in
all aspects of plant growth and development, including secondary
metabolism (e.g., see Payne et al., 1991).
[0068] The inventors have found that certain gas concentration
regimes may favor growth and secondary metabolism in cell cultures.
For example, a range of oxygen concentrations may be compatible
with culture cultivation, from 1% of air saturation to up to 200%
of air saturation, and preferably in the range of 10% to 100%, and
most preferably in the range of 25% to 95%. A range of carbon
dioxide concentrations may be compatible with culture cultivation,
from 0.03% (v/v in the gas phase that is in equilibrium with the
culture medium) to 15% (v/v), and preferably in the range of 0.3%
to 8% (v/v). The optimal concentrations of dissolved gases may
differ with respect to the cell metabolism, for example, cells
undergoing rapid growth may have different optima than cells
undergoing taxane biosynthesis, which typically favor higher oxygen
levels, and are less sensitive to higher carbon dioxide levels. The
optima may also vary with the kinetics of the culture; for example,
cells in the lag phase may prefer different dissolved gas
concentrations than cells in the logarithmic growth phase.
[0069] Dissolved gases may interact with other culture components
and with the action of enhancement agents in many ways. For
example, oxygen requirements may change upon elicitation or
stimulation of biosynthesis. Increases in respiration rates as a
wound response are commonly observed when plant cell cultures are
elicited. Elicitors or stimulators may mediate their action via
ethylene, or may affect ethylene production independently of
promoting secondary metabolism. In such cases, it may be desirable
to substitute a microbial elicitor preparation with ethylene, and
perhaps prevent toxicity associated with other microbial components
in the elicitor preparation. Alternatively, it may be advantageous
to inhibit the action of ethylene, thereby allowing the elicitor or
stimulant to promote secondary metabolism in a more exclusive, and
thereby more effective, manner. As described below, silver ion, a
component known to affect ethylene action, does advantageously
modify taxane biosynthesis.
Enhancement Agents
[0070] Production of secondary metabolites is a complex process,
requiring coordinated action of many different enzymes to produce
and sequentially modify the precursors which are ultimately
converted into the secondary metabolites. At the same time,
secondary metabolite production will be lowered if other enzymes
metabolize precursors of the desired metabolite, draining the
precursor pools needed to build the secondary metabolites.
[0071] Limitation of the amount of available precursor, due to low
production or subsequent diversion, or limitation in the conversion
of a precursor or intermediate to a downstream intermediate, or
limitation in the activity of a given enzyme, will limit the
production of secondary metabolites. In any particular culture
system, the rate at which a secondary metabolite is produced will
be controlled by one of these limitations, forming a bottleneck in
the pathway by which the precursor(s) are converted into the
secondary metabolite. Relieving the limitation which causes the
bottleneck will increase the rate of secondary metabolite
production in that culture system up to the point at which another
step in the pathway becomes limiting. The particular step which
limits the overall rate of production will vary between different
cultures, as will the action which relieves the limitation.
[0072] Taxanes are secondary metabolites which are produced through
a series of many enzymatic steps, and the present inventors have
determined several classes of enhancement agents which relieve one
or more of the rate limiting steps in taxane biosynthesis. Addition
of one of these enhancement agents to a culture of taxane-producing
cells will enhance the rate of taxane production. Furthermore, the
inventors have determined that use of the enhancement agents
discussed herein will have at least some enhancing effect in most
taxane-producing cultures, suggesting that the overall production
rate is determined not by a single rate-limiting step, but by a
complex interaction among a multiplicity of limiting factors.
Relief of any one of the limiting factors will enhance taxane
production, although the magnitude of the enhancement will depend
on particular culture conditions which determine the relative
limiting effects of other steps in taxane biosynthesis, once a
particular limitation has been relieved. Culture conditions which
affect the interaction between various limiting factors include the
genetic make up of the cells, the composition of the culture medium
and the gaseous environment, temperature, illumination and process
protocol, and the enhancement agent(s) added to a particular
culture will usually be selected in view of the limiting factors in
that culture, which may be determined empirically by comparing the
effects of individual enhancement agents as set forth herein.
Furthermore, it has been discovered that further enhancement of
taxane production will be achieved if more than one enhancement
agent is present in the culture.
[0073] Representative enhancement agents within the contemplation
of this invention are exemplified in Table 1. The enhancement
agents of this invention will be discussed under several general
classes. These classes are: anti-browning agents, anti-senescence
agents, anti-ethylene agents, plant growth regulators, such as
auxin-related growth regulators, precursors, inhibitors, elicitors,
stimulants and jasmonate-related compounds.
[0074] One class of enhancement agents contemplated by this
invention are anti-browning agents. As used herein, the term
"anti-browning agents" refer to components that are added to the
nutrient medium to prevent the formation of pigments during cell
cultivation. These pigments include phenolics and related compounds
that are generally observed to have a deleterious effect on cell
growth, viability, and product formation. A typical anti-browning
agent used in the nutrient media according to this invention is
ascorbic acid. Anti-browning agents may be typically incorporated
in the medium at a concentration range of 10 ppb to 1000 ppm.
[0075] Another class of enhancement agents is anti-senescence
agents. An anti-senescence agent is a compound of biological or
non-biological origin that protects cells from senescence. Such
agents could act by, for example, blocking the production of
compounds that promote senescence, blocking the action of
senescence-promoting factors, providing radical-scavenging or
anti-oxidant activities, protecting the integrity of cellular
membranes and organelles, or by other mechanisms. Such agents
include antagonists of ethylene action; polyamines and their
metabolites, such as spermine, spermidine, diaminopropane, and the
like; anti-browning agents, inhibitors of phenolics production, and
radical scavengers, such as reduced glutathione, propyl gallate,
and sulfhydryl compounds such as .beta.-mercaptoethanolamine.
[0076] Anti-ethylene agents are defined as substances that
interfere with ethylene production or ethylene action.
Anti-ethylene agents that interfere with ethylene Metabolism may be
further classified as ethylene-biosynthesis antagonists, and
ethylene-action antagonists. Ethylene-biosynthesis antagonists are
compounds that interfere with the biosynthetic pathway to ethylene;
examples of enzymes along this biosynthetic pathway that are
inhibited include ACC synthase, ACC oxidase, and ethylene oxidase.
Examples of ethylene biosynthesis antagonists include
.alpha.-aminoisobutyric acid, acetylsalicylic acid,
methoxyvinylglycine, aminooxyacetic acid and the like.
[0077] Examples of ethylene action antagonists include silver
containing compounds, silver complexes, or silver ions, carbon
dioxide, 1-methylcyclopropene, 2,5-norbornadiene,
trans-cyclooctene, cis-butene, diazo-cyclopentadiene and the like.
Suitable silver salts include silver nitrate, silver thiosulfate,
silver phosphate, silver benzoate, silver sulfate, silver salt of
toluenesulfonic acid, silver chloride, silver oxide, silver
acetate, silver pentafluoropropionate, silver cyanate, silver salt
of lactic acid, silver hexafluorophosphate, silver nitrite, and the
trisilver salt of citric acid. Illustrative examples of the
enhancement of taxane biosynthesis by a variety of silver salts are
shown in Example 10.
[0078] Anti-ethylene agents may be incorporated into the medium at
levels of 10 ppb to 1000 ppm. When silver is incorporated in the
medium, it will be added at a concentration of less than 900 .mu.M,
preferably less than 500 .mu.M, and more preferably less than 200
.mu.M. When silver is incorporated in the medium, it will be added
at a concentration of at least 10 nM, preferably 100 nM, more
preferably 1 .mu.M, and typically at 10 .mu.M.
[0079] Enhancement agents contemplated in this invention include
plant growth regulators, particularly auxin-related growth
regulators, which will include auxins, compounds with auxin-like
activity, and auxin antagonists. Auxin-related growth regulators
will typically be incorporated in the medium at concentrations of
between 10.sup.-10 M and 10.sup.-3 M, preferably between 10.sup.-8
and 10.sup.-5 M. Most preferred examples of auxin-related growth
regulators include 1-Naphthaleneacetic acid, 2-Naphthaleneacetic
acid, 1-Naphthaleneacetamide/Naphthylacetamide,
N-(1-Naphthyl)phthalamic acid, 1-Naphthoxyacetic acid,
2-Naphthoxyacetic acid, beta-Naphthoxyacetic acid,
1-Naphthoxyacetamide, 3-Chlorophenoxyacetic acid,
4-Chlorophenoxyacetic acid, 3-Iodophenoxyacetic acid,
Indoleacetamide, Indoleacetic acid, Indoylacetate, Indoleacetyl
leucine, Gamma-(3-Indole)butyric acid,
4-Amino-3,5,6-trichloropicolinic acid,
4-Amino-3,5,6-trichloropicolinic acid methyl ester,
3,6-Dichloro-o-anisic acid, 3,7-Dichloro-8-quinolinecarboxylic
acid, Phenylacetic acid, 2-Iodophenylacetic acid,
3-Iodophenylacetic acid, 2-Methoxyphenylacetic acid, Chlorpropham,
4-chloroindole-3-acetic acid, 5-Chloroindole-3-acetic acid,
5-Bromo-4-chloro-3-indoyl butyrate, Indoleacetyl phenylalanine,
Indoleacetyl glycine, Indoleacetyl alanine, 4-chloroindole,
p-chlorophenoxyisobutyric acid, 1-pyrenoxylbenzoic acid,
Lysophosphatidic acid, 1-naphthyl-N-methylcarbamate, and
Ethyl-5-chloro-1H-Indazole-3-ylacetate-3-Indolebutanoic acid. Other
preferred examples of auxin-related growth regulators include
Naphthalene-2,6-dicarboxylic acid,
Naphthalene-1,4,5,8-tetracarboxylic acid dianhydride,
Naphathalene-2-sulfonamide, 4-Amino-3,6-disulfo-1,8-naphthalic
anhydride, 3,5-dimethylphenoxyacetic acid, 1,8-Naphthalimide,
2,4-Dichlorophenoxyacetic acid, 2,3-Dichlorophenoxyacetic acid,
2,3,5-Trichlorophenoxyacetic acid, 2-Methyl-4-chlorophenoxyacetic
acid, Nitrophenoxyacetic acids,
DL-alpha-(2,4-Dichlorophenoxy)propionic acid,
D-alpha-(2,4-Dichlorophenoxy)propionic acid, 4-Bromophenoxyacetic
acid, 4-Fluorophenoxyacetic acid, 2-Hydroxyphenoxyacetic acid,
5-Chloroindole, 6-Chloro-3-indoylacetate, 5-Fluoroindole,
5-Chloroindole-2-carboxylic acid, 3-Chloroindole-2-carboxylic acid,
Indole-3-pyruvic acid, 5-Bromo-4-chloro-3-indoylbutyrate,
6-Chloro-3-indoylbutyrate, Quinoline-2-thioglycolic acid,
Aminophenylacetic acids, 3-Nitrophenylacetic acid,
3-Chloro-4-hydroxybenzoic acid, Chlorflurenol, 6-Chloro-3-indoyl
acetate, N-(6-aminohexyl)-5-chloro-1-Naphthalenesulfonamide
hydrochloride, 2-chloro-3(2,3-dichlorophenyl)propionitrile,
o-chlorophenoxyacetic acid,
6,7-dimethoxy-1,2-benzisoxazole-3-acetic acid,
3-oxo-1,2,-benzisothiazoline-2-ylacetic acid, Mastoparan,
2,3,5-Triidobenzoic acid, 2-(3-chlorophenoxy)propanoic acid, and
Mecoprop. Other examples of suitable auxin-related growth
regulators include Naphthoic acid hydrazide,
2,4-Dibromophenoxyacetic acid, 3-Trifluoromethylphenoxyacetic acid,
Oxindole, Indole-2-carboxylic acid, Indole-3-lactic acid,
Beta-(3-Indole)propionic acid, 2-Bromophenylacetic acid,
3-Bromophenylacetic acid, 2-Chlorophenylacetic acid,
3-Chlorophenylacetic acid, 2-Methylphenylacetic acid,
3-Methylphenylacetic acid, 3-Trifluoromethylphenylacetic acid,
3-Methylthiophenylacetic acid, Phenylpropionic acid,
4-chloro-2-methylphenylthioacetic acid, 2-Chlorobenzoic acid,
3-Chlorobenzoic acid, 2,3-Dichlorobenzoic acid, 3,4-Dichlorobenzoic
acid, 2,3,5-Trichlorobenzoic acid, 2,4,6-Trichlorobenzoic acid,
2-Benzothiazoleoxyacetic acid,
2-Chloro-3-(2,3-dichlorophenyl)propionitrile,
2,4-Diamino-s-triazine, Naphthalic anhydride, Dikegulac,
chlorflurecolmethyl ester, 2-(p-chlorophenoxy)-2-methylpropionic
acid, 2-chloro-9-hydroxyfluorene-9-carboxylic acid,
2,4,6-trichlorophenoxyacetic acid, 2-(p-chlorophenoxy)-2-methyl
propionic acid, Ethyl 4-(chloro-o-tolyloxy)butyrate,
[N-(1,3-dimethyl-1H-Pyrazol-5-yl)-2-(3,5,6-Trichloro-2-pyridinyl)oxy]acet-
amide, 4-Chloro-2-oxobenzothiazolin-3-yl-acetic acid,
2-(2,4-Dichlorophenoxy)propanoic acid,
2-(2,4,5-Trichlorophenoxy)propanoic acid, 4-Fluorophenylacetic
acid, 3-Hydroxyphenylacetic acid, Orthonil,
3,4,5-Trimethoxycinnamic acid, 2(3,4-dichlorophenoxy)triethylamine,
Indole-3-propionic acid, Sodium Ioxynil, 2-Benzothiazoleacetic
acid, and (3-phenyl-1,2,4-thiadiazol-5-yl)thioacetic acid.
[0080] Other classes of plant growth regulators may als be
incorporated into the nutrient medium as enhancement agents. These
include cytokinin-related growth regulators such as
N.sup.6-benzyladenine,
6-[.gamma.,.gamma.-dimethylallylamino]purine, kinetin, zeatin,
N-phenyl-N'-1,2,3-thidiazol-5-ylurea (thidiazuron) and related
phenylurea derivatives and the like, gibberrellins such as
GA.sub.3, GA.sub.4, GA.sub.7, and GA derivatives, abscisic acid and
its derivatives, brassinosteroids, and ethylene-related growth
regulators. Such growth regulators may be incorporated in the
medium at concentrations between 10.sup.-10 M and 10.sup.-3M,
preferable between 10.sup.-8 M and 10.sup.-5M.
[0081] Another class of enhancement agents are precursors or
biosynthetic precursors. As used herein, the term precursors are
used to describe compounds added to the nutrient medium that are
metabolized and incorporated by the cells into taxol and taxanes.
Suitable precursors include precursors of isoprenoid compounds such
as acetate, pyruvate and the like; .alpha.-phenylalanine,
.beta.-phenylalanine (3-amino-3-phenylpropionic acid),
phenylisoserine, N-benzoylphenylisoserine, benzoic acid, shikimic
acid, glutamine, cinnamic acid, and the like. Derivatives of the
aforementioned molecules are also suitable as precursors.
[0082] Another class of enhancement agents are inhibitors.
Inhibitors are compounds which inhibit enzymatic or other cellular
activities As used herein, the term "metabolic inhibitors" are used
to describe compounds added to the nutrient medium that interfere
with specific biosynthetic pathways. For example, a metabolic
inhibitor may be used to enhance taxol, baccatin III, or other
taxane biosynthesis by blocking a different pathway that competes
for an early biosynthetic precursor. Particularly effective
enhancement agents of this class include inhibitors of
phenylpropanoid metabolism, which are compounds capable of
inhibiting the synthesis or metabolism of cinnamic acid or its
derivatives. These compounds include preferably p-Coumaric acid,
4-Fluoro-DL-tyrosine, 4-Methoxybenzoic acid, 3-dimethylaminobenzoic
acid, 4-methoxycinnanic acid, 4-nitrocinnamic acid ethyl ester,
4-Nitrocinnamaldehyde, Mercaptoethanol, 4-hydroxycoumarin,
Cinnamylfluorene, 2-cyano-4-hydroxycinnamic acid,
Cinnamylidenemalonic acid, 4-dimethylaminocinnamic acid,
N-cinnamylpiperazine, N-Trans-cinnamoylimidazole,
2-Aminoindan-2-Phosphonic acid, Benzylhydroxylamine, Procaine,
Monensin, N-(4-Hydroxyphenyl)glycine, 3-(4-hydroxyphenyl)propionic
acid, 3-(2-hydroxyphenyl)propionic acid, more preferably
D-Phenylalanine, N-(2-mercaptopropionyl)glycine and its acetic acid
salt complex, DL-Metafluorophenylalanine,
p-Fluoro-DL-phenylalanine, Dithiothreitol, 4-Fluorocinnamic acid,
Trans-3,4-Difluorocinnamic acid, 3,4-Difluoro-D-Phenylalanine,
diethyldithiocarbamic acid,
4-Fluoro-(1-amino-2-phenylethyl)phosphonic acid,
3,4-methylenedioxybenzoic acid, and most preferably
3,4-methylenedioxy-6-nitrocinnamic acid, 3,4-methylenedioxycinnamic
acid, 3-[3,4-methylenedioxyphenyl]propionic acid,
3,4-methylenedioxyphenylacetic acid, 4-Fluoro-L-Phenylalanine,
4-Hydroxyphenylpyruvic acid, 4-Fluoro-DL-Tyrosine, Trans
3,4-Dimethoxycinnamic acid, phenylpropiolic acid,
L-2-Hydroxy-3-Phenylpropionic acid, 2-hydroxy-4,6-dimethoxybenzoic
acid, SKF-525A (2-(diethylamino) ethyl ester of
.alpha.-phenyl-.alpha.-propylbenzeneacetic acid), vinylimidazole,
ammonium oxalate, sinapic acid, and 1-aminobenzotriazole and
related analogs. When incorporated into the medium, the inhibitors
will be added at a concentration between 10 ppb and 1000 ppm,
preferably at a concentration between 100 ppb and 100 ppm, and more
preferably at a concentration of 1 ppm to 50 ppm.
[0083] In order to improve the yield of taxol, baccatin III, and
other related taxanes in cell cultures, the inventors have
undertaken a number of approaches. One of the approaches that has
been used to enhance productivity is the use of so-called
elicitors. As used herein, the term elicitors is used for compounds
of biological and non-biological origin that cause an increase in
secondary metabolite production when applied to plants or
plant-cell cultures (Eilert 1987, "Cell Culture and Somatic
Genetics of Plants," Vol. 4, F. Constabel and I. K. Vasil (eds.),
Academic Press, New York, pp. 153-196; Ebel, 1984, Bioregulators:
Chemistry and Uses. 257-271; and Darvill et al., 1984, Ann. Rev.
Plant Physiol., 35, 243-275). Many different compounds can act as
elicitors, depending upon their nature of origin and their mode of
action with cell metabolism. In these studies, the inventors have
used two major kinds of elicitors: 1) Biotic elicitors which
usually comprise cell wall extracts or filtrates from a selected
group of fungi, bacteria and yeasts, and also their purified
fractions. 2) Abiotic elicitors which have included chemical stress
agents as well as some compounds of biological origin (see
elicitors listed in Table 1). In addition, salts and complexes
containing heavy metal ions may also be considered as effective
abiotic elicitors; these include examples such as cobalt, nickel,
lanthanum, selenium, vanadium, lead, cadmium, chromium, aluminium,
iodine, barium, bismuth, lithium, rubidium, strontium, and gold. It
should be noted that certain compounds that mediate elicitation,
for example, the jasmonate-related compounds described below, may
also be considered as elicitors.
[0084] Christen et al. (1991) report the use of fungal elicitors
and selected compounds for production of taxol by suspensions of
Taxus brevifolia; however, the increases in the level of taxol
accumulation due to elicitor treatments have not been
specified.
[0085] In general, both kinds of elicitors were effective, although
the extent to which elicitation (taxane accumulation in cell
cultures as well as their secretion into the medium) occurred
differed from elicitor to elicitor and from species to species. The
highest production increase was attained with chitosan glutamate,
lichenan, ferulic acid and benzoic acid. Chitosan and lichenan are
complex polysaccharides derived from microbial cell walls. Chitosan
when used alone is insoluble in medium, and is toxic and causes
permanent cell damage. Chitosan glutamate, on the other hand, is
readily soluble in medium and does not affect cell viability.
Ferulic and benzoic acids are synthesized chemicals of biological
origin, and are generally used as anti-oxidants in biological
systems.
[0086] Elicitors and metabolic stress agents may be utilized
according to this invention to maximize taxol, baccatin III, and
total taxane production and secretion in tissue culture by
assessing elicitor specificity and concentration, timing, and
duration, as a function of culture age and media composition.
[0087] Another class of enhancement agents contemplated in this
invention are stimulants. As used herein the term stimulant is used
to describe compounds added to the nutrient medium that stimulate
or activate specific biosynthetic pathways, for example those
leading to biosynthesis.
[0088] Jasmonate-related compounds are a class of compounds that
mediate the elicitation reaction, thereby stimulating secondary
metabolite biosynthesis. Jasmonate-related compounds include
jasmonic acid and its alkyl esters, such as methyl jasmonate, ethyl
jasmonate, propyl jasmonate, butyl jasmonate, pentyl jasmonate,
hexyl jasmonate; dihydrojasmonic acid and its alkyl esters, such as
methyl dihydrojasmonate, ethyl dihydrojasmonate, n-propyl
dihydrojasmonate, butyl dihydrojasmonate, pentyl dihydrojasmonate,
hexyl dihydrojasmonate; epimethyl jasmonate, fluoromethyl
jasmonate, cis-jasmone, isojasmone, tetrahydrojasmone,
12-oxophytodienoic acid, dihydrojasmone, jasmonyl acetate,
apritone, amylcyclopentenone, hexylcyclopentenone,
hexylcyclopentanone, and related derivatives and analogs.
Jasmonate-related compounds are incorporated into the medium at
concentrations of 10.sup.-9 M to 10.sup.-3 M and preferably at
concentrations of 10.sup.-6 to 5.times.10.sup.-4 M, and more
preferably at concentrations of 10.sup.-5 M to 2.times.10.sup.-4 M.
It should be noted that more than one jasmonate-related compound
may be incorporated into the nutrient medium. It will be recognized
by the skilled artisan that the concentration of enhancement agents
such as jasmonate-related compounds, auxin-related growth
regulators, precursors, and other nutrients will change as these
compounds are metabolized in the culture. Unless otherwise
indicated, the concentrations recited herein refer to the initial
concentration in the nutrient medium.
[0089] Combining enhancement agents from at least two of the
following classes of enhancement agents has been shown to enhance
taxane production by Taxus cells beyond the maximum enhancement
observed for any one of the agents when used alone. These classes
of enhancement agents are elicitors, jasmonate-related compounds,
inhibitors of ethylene action, inhibitors of phenylpropanoid
metabolism, antisenescence agents, precursors and auxin-related
growth regulators. Therefore, in a preferred mode, this invention
provides methods for enhancing production of one or more taxanes by
culturing cells of a Taxus species in the presence of enhancement
agents selected from at least two of these agent groups.
[0090] Preferred methods for taxane production use the prototype
inhibitor of ethylene action, silver, in combination with at least
one other enhancement agent, and in particularly preferred methods
the other agent is methyl jasmonate, or an inhibitor of
phenylpropanoid metabolism, such as 3,4-methylenedioxynitrocinnamic
acid.
[0091] When used in combination with each other, jasmonate-related
compounds and ethylene-action inhibitors may be incorporated into
the nutrient medium in certain proportions to each other. For
example, when methyl jasmonate and silver thiosulfate are used in
combination, the molar ratios of methyl jasmonate to the silver ion
may be in the range between 0.0001 to 9.5, preferably in the range
between 0.001 to 8, more preferably in the range between 0.1 to 7,
and most preferably in the range between 1 to 5.
[0092] When used in combination with each other, auxin-related
growth regulators and ethylene-action inhibitors may be
incorporated into the nutrient medium in certain proportions to
each other. For example, when an auxin-related growth regulator and
silver thiosulfate are used in combination, the molar ratios of
auxin-related growth regulator to silver ion may be in the range
between 0.011 to 1000, preferably in the range between 0.015 to
100, and more preferably in the range between 0.02 to 50, and most
preferably between 0.05 to 30.
[0093] Generally, when culturing of Taxus cells for the production
of taxanes, one or more auxin-related growth regulator will be
added to the culture medium. Presence of auxin-related growth
regulator(s) will promote cell growth, but more significantly will
enhance production of taxanes by the culture. Further enhancement
can be obtained by adding at least one other enhancement agent
contemporaneously with the auxin-related growth factor.
[0094] In a preferred mode of this invention, one or more
enhancement agents are added to the culture in an amount sufficient
to enhance the production of one or more taxanes by at least
3-fold, preferably by at least 5-fold, more preferably by at least
10-fold, and even more preferably by at least 30-fold relative to
the level of production in the absence of the enhancer(s). In
another preferred mode of this invention, one or more enhancement
agents are added to the culture in an amount sufficient to enhance
the volumetric productivity of taxol at least to 10 mg/L/day, more
preferably to at least 15 mg/L/day, and even more preferably to at
least 22 mg/L/day. In another preferred mode of this invention, one
or more enhancement agents are added to the culture in an amount
sufficient to enhance the whole broth titer of taxol to at least
150 mg/L, more preferably to at least 200 mg/L, and even more
preferably to at least 350 mg/L. In another preferred mode of this
invention, one or more enhancement agents are added to the culture
in an amount sufficient to enhance the volumetric productivity of
baccatin III to at least 15 mg/L/day, more preferably to at least
20 mg/L/day, and even more preferably to at least 25 mg/L/day. In
another preferred mode of this invention, one or more enhancement
agents are added to the culture in an amount sufficient to enhance
the whole broth titer of baccatin III to at least 100 mg/L, more
preferably to at least 150 mg/L, and even more preferably to at
least 250 mg/L. In another preferred mode of this invention, one or
more enhancement agents are added to the culture in an amount
sufficient to enhance the volumetric productivity of taxanes to at
least 15 mg/L/day, more preferably to at least 25 mg/L/day, and
even more preferably to at least 40 mg/L/day. In another preferred
mode of this invention, one or more enhancement agents are added to
the culture in an amount sufficient to enhance the whole broth
titer of taxanes to at least 200 mg/L, more preferably to at least
300 mg/L, and even more preferably to at least 400 mg/L.
[0095] Many of the compounds described as enhancement agents above
have been used in other plant systems. Formulation, administration,
and appropriate physiological concentration levels in these
non-Taxus systems will provide guidance for the skilled artisan to
apply these agents in accordance with this invention.
Cellular Material
[0096] Suitable cells for culture in the method of this invention
may be from any species of Taxus. Preferably, the cells will be
from a cell line that inherently produces taxanes in relatively
high yield. Typically, such cells have the ability to produce high
levels of one or more taxanes under standard conditions or exhibit
high average volumetric productivities of taxanes under standard
conditions. Suitable cell lines may be identified by culturing
cells of the cell line under standard taxane production conditions
and observing the level of one or more taxanes produced in the
culture or determining the average volumetric productivity for one
or more taxanes by the in the culture the following procedures.
[0097] Cells for use in the production culture testing procedure
are grown in a suitable medium adapted for the particular cell
line. Following completion of log phase growth, an aliquot of cells
are cultured for test production of taxanes. Production culture is
generally performed in liquid medium, although callus culture on
solid medium may be used. In production culture, the cells are
cultivated in medium N from Table 2, in medium N from Table 2
except for replacement of sucrose by 7% (w/v) maltose, or in a
nutrient medium optimized for growth and maintenance of the
particular cell line. In the production culture, the cell density
should be in the range of 15-20 percent (w/v) on a fresh weight
basis. Cells are cultured for 10-20 days at 25 C+/-2 C under dark
conditions. Liquid cultures should be appropriately agitated and
aerated, for example on a rotary shaker at 120-180 rpm.
[0098] Production cultures for evaluating cell line characteristics
will include suitable enhancement agents. Generally, six
alternative enhancement cocktails (combinations of up to five
enhancement agents) are tested for each cell line. The combinations
are shown in Table A below.
[0099] At the end of the culture, titer of individual taxanes in
the culture may be measured by ELISA assay performed as described
herein, or the profile of taxanes produced in the culture may be
determined by HPLC analysis as described in Example 5. Preferred
cell lines will produce one or more taxanes above the minimum
target taxane levels in one or more of the enhancement cocktails.
Preferred cell lines will exceed the target levels for both titre
and productivity for at least one enhancement cocktail, and more
preferably for two or more enhancement cocktails. Minimum target
taxane titer at the end of production culture for suitable cell
lines will be at least 100 mg/L taxanes. Alternatively, the minimum
average volumetric productivity target over the course of the
production culture will be 10 mg/L/day taxanes. More preferred cell
lines will achieve minimum taxane titer at the end of production
culture of at least 100 mg/L taxol or 200 mg/L baccatin III, or
average volumetric productivity over the course of the production
culture of 10 mg/L/day taxol or 15 mg/L/day baccatin III.
TABLE-US-00001 TABLE A Enhancement Cocktails Combinations of
Enhancement Agents: 1. 20 .mu.M Naa + 30 .mu.M Mdna 2. 20 .mu.M Naa
+ 30 .mu.M Mdna + 50 .mu.M Slts 3. 20 .mu.M Naa + 30 .mu.M Mdna +
89 .mu.M Mjs 4. 20 .mu.M Naa + 30 .mu.M Mdna + 89 .mu.M Mjs + 50
.mu.M Slts 5. 20 .mu.M Naa + 30 .mu.M Mdna + 89 .mu.M Mjs + 50
.mu.M Slts + 5 mM Gln 6. 20 .mu.M Naa + 89 .mu.M Mjs + 50 .mu.M
Slts Gln = glutamine Naa = 1-naphthaleneacetic acid Mdna =
3,4-methylenedioxy-6-nitrocinnamic acid Mjs = methyl jasmonate Slts
= silver thiosulfate
[0100] Suitable production media for the various species are listed
in Table 5, although others may be used. For example, Media B, C
and N from Table 2 are particularly suitable production media for
Taxus chinensis. Media preferably contain the ingredients listed in
Table 2. These media preferably contain major and minor inorganic
salts, organics and growth hormones or growth regulators, in the
amounts generally with the preferred ranges starting with the
1/10th to three times the concentration of each medium ingredient
indicated in Table 2. Where medium B or N is used, the growth
regulators are typically incorporated into the medium in an amount
between 0.1 ppm to 20 ppm, and preferably between 1 ppm to 10 ppm.
When Medium C or N is used, the growth regulators are incorporated
preferably at levels ranging from 0.1 ppm to 5 ppm.
[0101] It will be understood by the skilled artisan that within the
contemplation of this invention modifications may be made in the
media described herein, such as substitution of other conventional
compositions (such as organics, vitamins, amino acids, precursors,
activators and inhibitors), addition or deletion of various
components, including growth regulators, or alteration of
proportions, so as to produce growth and taxane production equal to
or better than that observed with the media in Table 2.
Modes of Process Operation
[0102] The operating mode for a plant cell culture process refers
to the way that nutrients, cells and products are added or removed
with respect to time (Payne et al. 1991). When all the nutrients
are supplied initially, and the culture contents comprising cells
and product are harvested at the end of the culture period, the
operating mode is termed a "one-stage batch process". When a batch
process is divided into two sequential phases, a growth and a
production phase, with the medium being exchanged in between the
two phases, the operating mode is termed a "two-stage batch
process". Within the contemplation of this invention, the
transition from the growth medium through production medium, may
occur by an abrupt stepwise change, or progressively by a series of
continuous steps, or by progressive change. In one extreme the
progressive change is accomplished by progressive replacement of
medium, of incrementally changing composition. In another
alternative, the progressive change is accomplished by feeding one
or more components of the production medium into the growth phase
culture. This is one example of the fed-batch process.
[0103] In a "fed-batch" operation, particular medium components
such as nutrients and/or one or more enhancement agents are
supplied either periodically or continuously during the course of a
culture. It should be noted that certain components may be
incorporated into the nutrient medium initially in the batch mode,
then added in fed-batch mode, or may be added to the nutrient
medium exclusively in the fed-batch mode.
[0104] Using fed-batch operation, it has been found that cells can
be sustained in a productive state for a prolonged period, and in
fact, that productivity of the cells could be enhanced. As
illustrated in Examples 15 and 17, and in Tables 16 and 18, adding
certain nutrients and enhancement agents in a fed-batch manner gave
significant improvements in overall performance for taxanes
generally, and for specific taxanes such as taxol and bacctin III.
Further, this mode of operation has been found to be compatible
with a variety of different cell lines under a variety of different
media conditions.
[0105] Fed-batch addition of components is particularly
advantageous when the concentration of the particular component has
to be maintained at a low level in the culture, for example, to
circumvent the effects of substrate inhibition. Similarly,
fed-batch addition is advantageous when cells react negatively to a
component when it is either added initially to the nutrient medium
or if stoichiometrically-meaningful quantities of a component
cannot be added due to solubility or toxicity limitations. Further,
continuous or continual (periodic) fed-batch addition of a feed
solution containing a component is particularly preferred when
cells react negatively to the component when it is added in a more
rapid manner such as pulse addition. Particular components to which
cells respond favorable when added in a fed-batch mode include
taxane precursors such as alpha- and beta-phenylalanine; carbon
sources such as maltose, fructose and glucose; amino acids such as
glutamine, glutamic acid, aspartic acid; macronutrients such as
phosphate, calcium, and magnesuim; and enhancement agents such as
auxin-related growth regulators and jasmonate-related
compounds.
[0106] It will be apparent to the skilled artisan, that the
composition of the feed may be varied to obtain the desired results
such as extension of the production phase to increase taxane yield
or extension of the growth phase to achieve higher biomass density.
Selection of suitable conditions to achieve optimum productivity
and performance is easily within the skill of the ordinary artisan
in view of the teachings described herein. Similarly variations of
other operating parameters, such as the timing and duration of the
addition and the rate of the addition of the fed-batch components,
to achieve the desired results, are within the reach of the skilled
artisan in view of the teachings described herein.
[0107] Medium exchange as described herein refers to the removal of
spent medium from the culture followed by addition of fresh medium
to the culture; the cells are largely retained in the culture
during the operation. In the method of this invention, medium
exchange operation is an advantageous method to obtain and sustain
high volumetric productivities of taxane production, resulting in
superior process performance and overall production levels,
compared to a batch process. The extracellular product resulting
from such an operation may lend itself to more facile downstream
recovery and purification than other process modes.
[0108] As illustrated in Example 14 and Table 15, medium exchange
is successful in sustaining high productivities for taxanes
generally, and for specific taxanes such as taxol, baccatin III,
and 10-deacetylbaccatin III. In addition, this mode of operation
resulted in the increase in the volumetric productivity relative to
batch operation for taxanes generally, and for specific taxanes
such as taxol and baccatin III. Further, this mode of operation is
compatible with a variety of different cell lines under a variety
of different media conditions. As further illustrated in Example
7.3, the removal of spent medium and replenishment of fresh medium
every 3 days contributed to significant enhancement of taxane and
taxol production in growth conditions, as well as to an increase in
the amounts of extracellular product.
[0109] The stimulatory effects of medium exchange may have been due
to removal of product in situ, which would prevent feedback
inhibition and product degradation. Such positive effects of in
situ product removal on secondary metabolite production and
secretion in suspension cultures have been documented by, among
others, Robins and Rhodes (1986, Appl. Microbiol. Biotechnol., 24,
35-41) and Asada and Shuler (1989. Appl. Microbiol. Biotechnol.,
30, 475-481). The periodic removal of spent medium incorporates the
above advantages, and additionally, may serve to de-repress
secondary biosynthesis by removing other, non-taxane, inhibitory
components (such as phenolic compounds) from the medium.
[0110] The replenishment of fresh medium to cells undergoing active
biosynthesis may also enhance production by providing essential
nutrients that have been depleted. For example, Miyasaka et al.
(1986, Phytochemistry, 25, 637-640) were able to stimulate
stationary phase cells of Salvia miltiorhiza to produce the
diterpene metabolites, cryptotanshinone and ferruginol simply by
adding sucrose to the medium. Presumably, biosynthesis had ceased
due to carbon limitation in the stationary phase. The
periodic-medium-exchange protocol used in the present work could
have been beneficial as a result of any of the above factors. It is
understood that the amount of medium exchanged, the frequency of
exchange, and the composition of the medium being replenished may
be varied. The ability to stimulate biosynthesis and secretion by
medium exchange has important implications for the design and
operation of an efficient commercial process in the continuous,
semi-continuous or fed-batch mode.
[0111] When a substantial portion, but not all, of the contents of
a batch culture is harvested, with addition of fresh medium for
continued cell growth and production, the process resembles a
"repeated draw and fill" operation, and is termed a
"semi-continuous process". When fresh medium is continuously
supplied, and effluent medium is continuously removed, the process
is termed "continuous". If cells are retained within the reactor,
the process is termed a "perfusion mode" If cells are continuously
removed with the effluent medium, the continuous process is termed
a "chemostat".
[0112] It is understood that these various modes of process
operation are compatible with the taxane-production system
described herein.
EXAMPLES
[0113] The following examples are provided to further describe the
materials and methods which may be used in carrying out the
invention. The examples are intended to be illustrative and are not
intended to limit the invention in any manner.
Example 1
Callus Initiation
[0114] Samples of Taxus plant material were collected from a number
of wild and cultivated plants. Samples were processed upon arrival
at the laboratory or stored at 4.degree. C. until they could be
used.
[0115] The material was first washed in dilute soap solution,
rinsed in water, and the surface sterilized in a CLOROX solution
(1% hypochlorite, pH 7) for 10 minutes. Under sterile conditions
the material was then rinsed 3 times with sterile water. Needles
were then cut in a 1% polyvinylpyrrolidone (PVP) solution with 100
mg/l ascorbic acid. Needles were placed with the cut end in Medium
E (see Table 2). Thirty to forty explants were cultured per plate
of medium. Plates containing explants were incubated at
25.+-.1.degree. C. in the dark. Plates were monitored daily for the
appearance of contaminating micro-organisms, and where they were
present, uncontaminated needles were removed and placed in a fresh
plate of Medium E. Substantial callus formation was observed and
the callus was separated from the explant at 20 days and placed on
the various callus proliferation media listed in Table 3. For
example, calli of Taxus chinensis were transferred to Medium D (see
Table 2). This initiation procedure was very efficient, resulting
in low contamination rate and high frequency of callus induction of
over 90% of explants initiated. The same procedure was successfully
used to initiate cultures of Taxus brevifolia, Taxus canadensis,
Taxus cuspidata, Taxus baccata, Taxus globosa, Taxus floridana,
Taxus wallichiana, Taxus media, and Taxus chinensis.
Example 2
Callus Proliferation
[0116] Once calli were removed from the explant, they were
cultivated at 25.+-.1.degree. C. in the dark. Healthy parts of the
callus were transferred to fresh medium every 7 to 10 days, and
this frequency of transfer was found to be extremely important for
prevention of browning and for prolonged callus maintenance. The
preferred growth and maintenance media for calli of various species
are summarized in Table 3.
Example 3
Suspension Initiation
[0117] 1 g fresh weight of callus material was aseptically
inoculated into a 125 ml Erlenmeyer flask containing 25 ml of
liquid medium appropriate to each species (see Table 3). For
example, Medium D was used for Taxus chinensis. The flask was
covered with a silicone foam cap (Bellco, N.J.) and placed on a
gyratory shaker at 120 rpm at 24.+-.1.degree. C. in darkness.
Suspension cultures were formed in approximately 3 to 10 days.
Initially, medium was exchanged by suction filtering the flask
contents through a buchner funnel containing a miracloth filter
(Calbiochem), and resuspending all the biomass in fresh medium.
Upon cell growth, 1-2 g (fresh weight) of cells, and were generally
transferred into a new 125 ml flask containing 25 mL of fresh
medium and were thereafter subcultured weekly.
Example 4
Growth of Suspended Cells
[0118] The typical growth rates and cell densities achieved in
suspension cultures of representative species are listed in Table
4.
[0119] As a detailed example, the increase in biomass (fresh and
dry weight) with time for Taxus chinensis line K-1 is shown in FIG.
1. The maximum growth rate was measured by taking the slope at
points of most rapid biomass increase on the growth curves. Cell
cultures of Taxus chinensis grew at a maximum doubling time of 2.5
days. This growth rate is significantly higher than that reported
previously for Taxus species suspension cultures. For example,
Christen et al. (1991) reported a 5- to 10-fold increase in biomass
after 3 to 4 weeks of culture, which translates to an average
doubling time for Taxus brevifolia suspensions of 7 to 12 days.
[0120] The ability to cultivate cells at a high density is
important in maximizing the volumetric productivity of a cell
culture process. While cultures of Taxus brevifolia reached a cell
density of less than 1 g dry weight per liter (calculated from data
presented in Christen et al. (1991)), suspensions of Taxus
chinensis were able to reach densities of up to 8 to 20 g dry
weight per liter after 18 days of growth. The viability of cells
was determined by staining cells with a 0.05% solution of
fluorescein diacetate in acetone (Widholm, 1972, Stain Technol.,
47, 189-194), and by counting the number of green fluorescing cells
upon excitation with blue light in an inverted fluorescence
microscope (Olympus IMT-2, Japan). Cell viability was higher than
90% throughout the growth phase.
[0121] The ability to cultivate cells under rapid growth conditions
to high cell densities while retaining high viability is an
important pre-requisite to the economic operation of a plant cell
culture process for producing taxol, baccatin III, and taxanes.
Example 5
Analysis of Taxol, Baccatin III and Other Taxanes
5.1. ELISA Methods
[0122] ELISA analysis (Hawaii Biotech #TA-01) was used for
detection of taxol in cell culture extracts (see Grothaus, et al.,
1995). This method provides high sensitivity (0.1 ng/mL), however,
because a polyclonal antibody is used, cross-reactivity with other
taxanes is observed. Preparative (analytical scale) HPLC with
fraction collection showed cross-reactivity with 10-deacetyltaxol,
7-xylosyl-10-deacetyltaxol, cephalomannine, 10-deacetyl-7-epitaxol,
7 epitaxol, as well as other unidentified taxanes. Despite such
cross-reactivity this method was found to be extremely useful for
detection of taxane production and allowed large numbers of cell
lines to be screened quickly. Cell extracts showing significant
production of taxanes were then analyzed in detail using the HPLC
procedures outlined below.
[0123] A monoclonal ELISA analysis (Hawaii Biotech #TA-02) was also
used for detection of taxol in cell culture extracts. This method
provides high sensitivity (0.1 ng/mL) and significantly less
cross-reactivity.
5.2. Extraction of Taxol, Baccatin III, and Other Taxanes
[0124] Extraction of taxanes from supernatants were performed by
several methods depending on the concentrations present. When
sufficient amounts of taxanes (approx. 1-5 mg/L) are present in
liquid media, samples were prepared very rapidly and efficiently.
Media (2 mL) were dried completely (in vacuo) and a measured amount
of methanol (0.5-2.0 mL) was added. This mixture was agitated
ultrasonically until complete dissolution or dispersion of the
sample was accomplished. Solids were removed by centrifugation
prior to HPLC analysis. Quantitative recoveries have been obtained
at 1 mg/L levels with detection levels well below 0.1 mg/L.
[0125] When concentration of taxanes in the culture supernatants
were very low (less than 1 mg/L), the medium was extracted three
times with an equal volume of a mixture of methylene chloride and
isopropyl alcohol (IPA) (9:1 by vol.). The organic layer was
reduced to dryness and reconstituted in a measured volume of
methanol (50-250 mL). Multiple extraction typically recovered
90-95% of the taxol, cephalomannine, and baccatin III at 0.6 mg/L
levels.
[0126] When taxane concentrations in the supernatant exceeded
.about.5 mg/L a more rapid sample preparation was employed. One
part (vol.) of supernatant was mixed with 3 parts (vol.) of
methanol containing 0.1% acetic acid. This mixture then was
sonicated for 30 minutes, filtered, and analyzed by HPLC.
[0127] Samples of whole broth (culture supernatant containing
cells) were prepared using a method similar to that described in
the preceding paragraph. One part (vol.) of whole broth was mixed
with 3 parts (vol.) of methanol containing 0.1% acetic acid. This
mixture then was sonicated for 30 minutes, allowed to stand for an
additional 30 minutes, filtered and then analyzed by HPLC.
[0128] Cell materials were extracted by freezing freshly harvested
cells (-5.degree. C.), followed by vacuum drying, and methanol
soxhleting for 50 cycles. The volume of methanol was reduced
(.about.100 fold) by rotary evaporation and the resulting sample
was analyzed by HPLC. 70 to 80% of the taxanes were generally
recovered with 10-15% measurable decomposition. It was later found
that exhaustive drying of the sample prior to soxhlet resulted in
less than 5% degradation of taxol
[0129] The extraction of solid media and callus was accomplished
identically to that of cells when taxane levels were low, however,
methylene chloride/IPA vs. water partitioning of the final methanol
extract was always performed. When taxane levels exceeded .about.5
mg/L the whole broth extraction method was employed to prepare
samples of callus on solidified medium.
5.3. High Performance Liquid Chromatography Methods
[0130] Analytical high performance liquid chromatography (HPLC) was
performed on a high-carbon loaded diphenyl column (Supelco, 5 mM,
4.6 mm.times.25 cm) with an LDC Analytical binary gradient high
pressure mixing system consisting of CM3500/CM3200 pumps, a CM4100
variable volume autosampler and an SM5000 photo diode array
detector interfaced to a personal computer. Column temperature was
regulated at 35.degree. C. with an Eldex CH150 column oven.
Quantitative HPLC analysis of taxanes was accomplished using a
binary gradient elution scheme as follows:
TABLE-US-00002 Time % Eluant A % Eluant B Flow 0 75 25 1 mL/min 40
35 65 '' 42 25 75 '' 47 25 75 '' 50 75 25 '' Eluant A = 0.015 mM
KH.sub.2PO.sub.4 brought to pH 3.5 with trifluoroacetic acid Eluant
B = acetonitrile
[0131] The chromatographic methods used resemble several published
methods (Witherup et al. 1989, J. Liq. Chromatog., 12, 2117-2132)
with the exceptions that a phosphate buffer containing
trifluoroacetic acid has been used and that a longer gradient is
employed. These differences significantly improve the resolution of
taxol and other taxanes from the mixture. The relative retention
times observed for taxanes are shown below. Taxol elutes between 31
and 33 minutes depending on the column and hardware used.
TABLE-US-00003 Compound Relative Retention Time 10-deacetylbaccatin
III 0.38 baccatin III 0.56 7-xylosyl-10-deacetyltaxol 0.80
10-deacetyltaxol 0.87 cephalomannine 0.94 10-deacetyl-7-epitaxol
0.98 taxol 1.00 7-epitaxol 1.12
[0132] The retention times of taxol, cephalomannine and baccatin
III were determined using authentic samples obtained from the
National Cancer Institute. The retention times of the other taxanes
listed above were compared to analytical standards provided by
Hauser Chemical (Boulder, Colo.). Identification of known taxanes
was based on retention time and ultraviolet spectral comparisons.
Compounds that exhibited a UV spectrum similar to that of taxol and
baccatin III, but that did not correlate to the relative retention
times of these taxanes were considered taxanes. Quantitation of
taxol, cephalomannine and baccatin III was based on response
factors determined from authentic materials. Quantitation of
10-deacetylbaccatin III was performed using the response factor
determined for baccatin III. Where appropriate, quantitation of the
remaining taxanes was based on the response factors measured for
taxol and baccatin III. The term total taxanes represents the sum
of the taxanes that exhibited a characteristic UV similar to taxol
and baccatin III. Total taxanes identified in Taxus cultures
include, among others, 10-deacetylbaccatin III, 9-dihydrobaccatin
III, 7-epi-10-deacetylbaccatin III, baccatin III,
9-dihydro-13-acetylbaccatin III,
7-xylosyl-10-deacetylcephalomannine, 7-xylosyl-10-deacetyltaxol,
7-epibaccatin III, 10-deacetyltaxol, 7-xylosyltaxol,
cephalomannine, 7-epi-10-deacetyltaxol, taxol,
2-benzoyl-2-deacetyl-1-hydroxybaccatin I, taxol C, 7-epitaxol, and
2-benzoyl-2-deacetylbaccatin I.
[0133] Taxanes that did not exhibit the characteristic UV
absorbance, but did exhibit characteristics
taxane-mass-fragmentation characteristics upon mass spectrometry,
were also observed in Taxus cell cultures. Examples of such taxanes
produced in Taxus cell cultures are, among others, Taxuyunnanine C,
and its analogs and derivatives.
[0134] Each of the standards (10 uL) was typically injected
(initially then after 3 or 4 samples) and areas for each of the
three components were integrated from the 227 nm chromatogram.
Response factors for each of the components was obtained by linear
least-squares analysis of the data. 10 uL of each sample was
injected and the amount per injection was calculated based on the
standard data regression. These results were converted to amount
per liter or percent dry weight. FIG. 4 illustrates a typical
chromatogram of a supernatant sample.
5.4 Rapid High Performance Liquid Chromatography Methods
[0135] In addition to the above method, several rapid methods of
HPLC analysis were developed to allow greater sample throughput.
Two of these methods are described in detail below.
[0136] Method 1). Rapid high performance liquid chromatography
(HPLC) was performed on a Phenomenex Curosil-G column (5 uM, 4.6
mm.times.25 cm with 4.6 mm.times.3 cm guard) at ambient temperature
using the hardware described above. Quantitative HPLC analysis of
taxanes was accomplished using a binary gradient elution scheme as
follows:
TABLE-US-00004 Time % Eluant A % Eluant B Flow 0 60 40 1.5 mL/min
10 25 75 '' 11 25 75 ''
[0137] Eluant A=0.01 mM KH.sub.2PO.sub.4 brought to pH 3.5 with
trifluoroacetic acid
[0138] Eluant B=acetonitrile
[0139] The relative retention times observed for taxanes are shown
below. Taxol elutes at about 8 minutes depending on the column and
hardware used.
TABLE-US-00005 Compound Relative Retention Time 10-deacetylbaccatin
III 0.42 baccatin III 0.61 taxol 1.00
[0140] Standards containing taxol, baccatin III and
10-deacetylbaccatin III were prepared at 50 mg/L, 10 mg/L, and 1
mg/L levels. A standard was injected initially and then after every
ninth sample and areas for each of the three components were
integrated from the 227 nm chromatogram. Response factors for each
of the components was obtained by linear least-squares analysis of
the data. 10 .mu.L of each sample was injected and the amount per
liter was calculated from the peak area based on the sample
dilution and the standard data regression.
[0141] Method 2). Rapid high performance liquid chromatography
(HPLC) was also performed on a Phenomenex IB-SIL Phenyl column (3
uM, 4.6 mm.times.15 cm with 4.6 mm.times.3 cm guard) at ambient
temperature using the hardware described above. Quantitative HPLC
analysis of taxanes was accomplished using a binary gradient
elution scheme as follows:
TABLE-US-00006 Time % Eluant A % Eluant B Flow 0 65 35 1.0 mL/min
10 30 70 '' 12 30 70 ''
[0142] Eluant A=0.015 mM KH.sub.2PO.sub.4 brought to pH 3.5 with
trifluoroacetic acid
[0143] Eluant B=acetonitrile
[0144] The relative retention times observed for taxanes are shown
below. Taxol elutes at about 9.5 minutes depending on the column
and hardware used.
TABLE-US-00007 Compound Relative Retention Time 10-deacetylbaccatin
III 0.41 baccatin III 0.61 taxol 1.00
[0145] Quantitation was performed as described above.
[0146] Modifications of the above methods with respect to flow rate
and gradient span and time were also found to perform suitable
chromatography for plant cell culture analysis.
5.4. MS/MS Confirmation of Taxol
[0147] The identity of taxol in cell culture supernatant has been
confirmed using an MS/MS method (as shown in FIG. 6) which couples
flow injection with ion spray atmospheric pressure chemical
ionization. Details of the procedures used for acquiring the data
presented in FIG. 6 were as follows: Mass Spectrometer: Sciex API 3
triple quadrupole with an atmospheric pressure ionization source.
Nitrogen was used as the curtain gas and argon was used as the
collision gas for the CID spectra. Interface: Ion Spray interface
producing ions by Ion Evaporation Ionization (Electrospray). Zero
air was used as the nebulizer gas. LC Pump: ABI 140B dual syringe
pump operating at 5 .mu.L/minute. Solvents: 50/50 acetonitrile/H2O
2 mM NH4OAc+0.1% formic acid. Injection Volume: 5 .mu.L, all
spectra taken by flow injection analysis. This method provided
unequivocal confirmation for the presence of taxol in cell culture
samples, and also provided quantitation with excellent agreement to
HPLC results.
Example 6
Taxol Production by Various Species
[0148] The taxol produced by cell cultures of various Taxus species
is summarized in Table 5. Callus was cultivated for 20 days in the
dark on the indicated solidified medium for each species. The cells
and medium were dried and methanol-extracted together, and assayed
by either ELISA or HPLC as indicated.
Example 7
7.1. Production in Growth Medium
[0149] The production of taxol and taxanes commenced within the
first 2 days of transfer into growth of Taxus chinensis cell line
K-1 into Medium A. The maximum taxol observed was on day 15, at
8.81 .mu.g/flask, which corresponds to 0.44 mg/liter taxol. Of
this, 46.1% was present in the extracellular medium. On day 15, the
total taxane concentration was 72.87 g/flask, or 3.6 mg/liter, of
which 58.6% was present in the extracellular medium. The viability
of cells was always greater than 90% as measured by fluorescence
staining (Example 4), suggesting that the presence of extracellular
taxol and taxanes was due to secretion rather than due to cell
lysis.
[0150] The production levels of taxol, baccatin III, and related
taxanes have been characterized for numerous different cell lines
under a number of different growth conditions (elaborated in Table
2 and in other examples) in which taxane biosynthesis is not
enhanced. These collective data indicate that when cultures are
cultivated under conditions optimized for growth, but not for
taxane biosynthesis, taxol production levels are typically less
than or equal to 0.5 mg/L, and always less than or equal to 2 mg/L;
the taxol volumetric productivities typically range from 0.03
mg/L/day to 0.07 mg/L/day, and are always less than 0.3 mg/L/day.
Similarly, baccatin III production levels are typically less than
or equal to 0.5 mg/L, and always less than or equal to 1 mg/L; the
baccatin III volumetric productivities are typically less than or
equal to 0.03 mg/L/day, and always less than 0.15 mg/L/day.
Similarly, total-taxane titers are typically less than 5 mg/L, and
are always less than or equal to 20 mg/L; the total taxane
volumetric productivities are typically less than 1 mg/L/day, and
always less than 3 mg/L/day.
7.3. Medium Exchange for Productivity Enhancement
[0151] Significant improvements in taxol and total taxane
productivity were obtained by aseptically suctioning off growth
Medium A on day 9, replacing with fresh medium and repeating the
procedure on day 12. The experiment was terminated on day 15, and
the results are shown in FIG. 2. The important increases in
productivity due to medium exchange are summarized in Table 6. The
total amounts of taxol and taxanes produced were ca. 4.6-fold
higher with medium exchange compared to controls without treatment.
Importantly, ca. 4.9-fold higher taxol, and ca. 5.9-fold higher
total taxanes were recovered in the extracellular medium compared
to controls without medium exchange treatment.
[0152] The ability to markedly enhance taxol and total taxane
productivities, and moreover, to cause extracellular product
accumulation is important for operation of an efficient, continuous
process with biomass reuse and simplified downstream
purification.
7.3. Effect of Light on Taxane Production in Growth Medium
[0153] Light is known to play an important role not only in
photosynthesis, but also in various aspects of secondary metabolism
in plant cell cultures (Seibert and Kadkade 1980). Whereas the
experiments described in Examples 4, 7.1, and 7.2 were conducted in
darkness, the response of Taxus chinensis cultures to light is
described here.
[0154] One gram fresh weight of 7-day old cells of Taxus chinensis
line K-1 were inoculated in 25 ml of growth Medium A (see Table 2)
in 125 ml Erlenmeyer flasks and incubated at 24.+-.1.degree. C. on
a gyratory shaker at 120 rpm. Duplicate flasks were placed in the
dark and under a Standard GroLux lamp at a distance of 3 feet.
Spectral characteristics of the lamp are shown in FIG. 3. Results
are shown in Table 7.
[0155] Exposure of cultures to light did not affect total taxane
levels or the extent of extracellular accumulation. However, taxane
profiles were significantly altered in the two treatments. For
example, cells cultivated in the light produced 2.8 fold higher
taxol than did cells in the dark. The proportion of extracellular
taxol was also significantly higher than in the dark treatment (76%
vs 56%). The use of light treatment, especially of specific
spectral quality, might be useful in a cell culture process for
taxol production.
Example 8
Elicitors
[0156] The term elicitors is used for compounds of biological (or
biotic) and non-biological (or abiotic) origin that cause an
increase in secondary metabolism when added to plant cell
cultures.
[0157] While a number of elicitors have been found useful, a
representative illustrative example is described here in detail,
namely, the use of chitosan glutamate. While chitosan has been
previously tried as an elicitor in some plant cell culture systems,
the accompanying toxic reactions such as browning and loss of
viability have made its use impractical (Beaumont and Knorr 1987,
Biotechnol. Lett. 9, 377-382). Indeed such toxic side reactions are
a common drawback of many elicitors reported in the literature. The
use of chemically modified chitosans such as chitosan glutamate to
specifically induce taxol and taxane biosynthesis while
circumventing toxic side-effects is a novel approach.
[0158] Suspensions of Taxus chinensis line K-1 grown in Medium D
for 7 to 8 days were suction filtered aseptically using a sterile
Buchner funnel fitted with a miracloth (Calbiochem) filter. 2 g
fresh weight cells were aseptically transferred to 25 ml of medium
C (see Table 2) in a 125-mL Erlenmeyer flask. A solution of 0.05%
chitosan glutamate was prepared freshly and filter-sterilized
through a 0.22 micron cartridge filter. 825 .mu.L of this solution
was added to the flask at the start of the experiment,
corresponding to a level of 165 mg elicitor per gram dry weight
cells. The flasks were incubated at 24.+-.1.degree. C. on a
gyratory shaker at 110 rpm in the dark. The flasks were
destructively sampled on day 15, and observations on growth, color
of the cells and medium and cell viability were recorded. Samples
were analyzed for taxanes as described in Example 5. The results of
this experiment are shown in Table 8.
[0159] Elicitor treatment resulted in a modest improvement in the
per-cell total taxane production (0.53% vs. 0.42% dry weight
taxanes) over non-treated controls. The non-toxic nature of the
elicitor is evident from the high viabilities (75-80%) observed in
both treatments. In fact, an increased dry weight in elicitor
treatment compared to controls has been reproducibly observed (14.2
g/l vs. 10.1 g/l dry weight). The higher cell densities resulted in
an 1.8-fold greater titer of total taxanes in the elicitor
treatment, i.e., 75.8 mg/L versus 42.4 mg/L for the control.
[0160] The elicitor treatment resulted in increased taxol
biosynthesis, both on a per-cell basis (0.098% vs. 0.054% dry
weight taxol, a 1.8-fold increase) and in a titer comparison (13.9
mg/L versus 5.4 mg/L, a 2.6-fold increase). The extent of secretion
was higher for the elicitor treatment compared to the control (85%
versus 72% extracellular product).
[0161] The elicitor treatment described herein results in increased
taxol production, a more favorable product profile, enhanced
product secretion and retention of high cell viability. These
production characteristics represent a significant improvement for
a cell culture process for taxol production.
Example 9
Production Medium Development
[0162] In an effort to increase taxol productivities over the
levels described in example 6, nutrient levels were manipulated to
formulate special `production media`. 7 to 8 day old suspensions of
Taxus chinensis line K-1 grown in Medium D were suction filtered
aseptically using a sterile Buchner funnel fitted with a MIRACLOTH
(rayon polyester cloth with acrylic binder) filter (Calbiochem).
500 mg fresh weight cells were aseptically transferred to 5 ml of
production Media B and C (see Table 2). The vessels were incubated
for varying time periods of 18, 25, and 42 days at 24.+-.1.degree.
C. on a gyratory shaker at 110 rpm in the dark. Treatments were
destructively sampled, and observations on growth, color of the
cells and medium, and cell viability were recorded. Samples were
analyzed for taxanes as described in Example 5. The results of this
experiment are shown in Table 8.
9.1. Results of 18-day Cultivation
[0163] Taxus chinensis cell cultures responded to the altered
medium compositions by producing significant levels of taxanes and
taxol. These data are summarized in Table 9, and a sample
chromatogram is shown in FIG. 4. In medium B, 99.8 mg/liter of
total taxanes were produced, with 24.1 mg/liter of taxol. In Medium
C, 110 mg/liter of total taxanes were produced, with 21.3 mg/liter
of taxol. On a dry weight basis, cells produced 0.18% dry weight
taxol on medium B, and 0.065% dry weight taxol on medium C.
9.2. Prolonged Cultivation
[0164] Taxol and taxane production after prolonged cultivation of
Taxus chinensis cells (line K-1) for 25 and 42 days was studied in
medium C, the results for which are summarized in FIG. 5. The
following significant observations can be summarized:
[0165] (i) Taxus suspension cultures are capable of producing
significant levels of taxol and other taxanes. Highest accumulation
occurred at 42 days, with 0.32% dry weight taxol, and 0.62% dry
weight total taxanes; corresponding to titers of 153 mg/L taxol and
295 mg/L total taxanes based on final medium volume. The analysis
of this sample by tandem mass spectrometry confirmed the presence
of taxol as shown in FIG. 6. Quantitation by MS/MS showed excellent
agreement with HPLC.
[0166] (ii) The rate of taxol biosynthesis between days 25 and 42
was at ca. 7.6 mg taxol per liter per day assuming linear
production in the 17-day period. This rate is significantly higher
than the rate of production in the first 25 days. The rate of total
taxane biosynthesis between days 25 and 42 was 12.3 mg per liter
per day. The average volumetric productivities for taxol, baccatin
III, and total taxanes were 3.6, 0.5, and 7.0 mg/L/day
respectively.
[0167] (iii) Production medium formulations can induce up to
45-fold increases in specific taxol content compared to
rapid-growth conditions (in which taxane biosynthesis is
unenhanced) such as those described in Example 7.
[0168] (iv) The product spectrum can be manipulated so as to funnel
biosynthesis towards the desired end-product taxol, while
minimizing production of undesirable taxanes. For example, on day
25, taxol constituted 28% of the total taxanes and on day 42, taxol
constituted 52% of the total taxanes in contrast to growth medium
(see Example 7.1), in which taxol constituted only 12.2% of the
total taxanes. This ability to manipulate product profiles will
have important repercussions for downstream purification and for
product purity-related regulatory issues. For example, the ability
to suppress production of the taxane by-product, cephalomannine
could greatly simplify downstream purification compared to
purification of taxol from bark tissue.
[0169] (v) Taxus cell cultures have been induced to secrete
significant amounts of taxol (87% on day 42) and other taxanes.
That the presence of extracellular taxol and taxanes is due to
secretion rather than due to cell lysis is corroborated by several
independent observations: (a) Continued biosynthesis occurred
between days 25 and 42, suggesting that cells were viable and
active. Independent observations have shown that >70% viability
have been observed after 18 days in production medium. (b)
Different percentages of different taxanes were secreted. If cells
had lysed, the percentage in the medium might have been expected to
be similar for the different taxanes.
[0170] (vi) The ability of this Taxus cell line to thrive and
produce taxol at high rates in an extracellular environment so rich
in product is particularly worth noting.
[0171] (vii) The Taxus cell line with which these results were
obtained is also capable of rapid growth to high cell densities,
and expressed the reported productivities after 20 generations
under rapid-growth conditions, attesting to its stability and
commercial potential.
[0172] The levels of taxol and taxanes produced by cell lines of
Taxus chinensis under the conditions described herein are higher
than previously reported results by a factor of 35- to 150-fold.
For example, Christen et al. (1991) reported the production of 1 to
3 mg/liter of taxol by suspension cultures of Taxus brevifolia
after 2 to 4 weeks of cultivation. Wickeramesinhe and Arteca (1991)
reported the production of taxol at 0.009% dry weight in cell
cultures of Taxus media.
[0173] In summary, our data show that with careful initiation and
selection of Taxus chinensis cultures, and with specially
formulated growth medium conditions, cells can be induced to grow
rapidly to high cell densities. When these cells are transferred to
production medium conditions, cells are able to biosynthesize and
secrete significant levels of taxol and other taxanes for prolonged
periods while maintaining high viabilities. The incorporation of
periodic medium exchange, light and elicitors with production
medium results in further synergistic productivity enhancements.
These properties are critical prerequisites for an efficient
commercial process for taxol and taxane production using tissue
culture technology.
Example 10
10.1. Enhancement of Taxane Production Using Silver
[0174] Silver, in the form of silver containing compounds, silver
complexes, or silver ions, was found to be a useful enhancement
agent of taxol, baccatin III, and taxane biosynthesis in cell
cultures of Taxus species. The combination of silver and other
enhancement agents has also been found to be useful in obtaining
and sustaining high rates of taxane production.
[0175] Seven-day old cells of Taxus chinensis suspension KS1A
cultivated in Medium L (Table 2) were suction filtered aseptically
using a sterile Buchner funnel fitted with a MIRACLOTH (Calbiochem)
filter. Approximately 0.75 to 1 gram fresh weight cells were
inoculated into 4 to 5 mL of culture medium of the given
composition indicated in Table 10, to yield a fresh weight cell
density in the range of 15% to 20% (w/v). The vessels were
incubated at 25.+-.1.degree. C. at 120 RPM on a gyratory shaker
(1'' throw) in the dark. Evaporation was corrected for by addition
of sterile distilled water. Samples of whole broth (i.e., both
extracellular and intracellular taxanes) were taken at periodic
intervals, and were processed and analyzed by HPLC according to the
methods outlined in Example 5.
[0176] The data summarized in Table 10 indicate that the production
of taxol, baccatin III, and other taxanes can be successfully
enhanced by a variety of silver containing compounds. This
enhancement is due primarily to the presence of silver in the
medium, as demonstrated in Table 10, which shows enhancement for a
variety of different silver containing compounds and different
counterions. These levels of production are significantly higher
than that observed in unenhanced cultures (the production levels
for which are elaborated in Example 7).
10.2. Enhancement of Taxane Production Using Silver Thiosulfate
[0177] Based on considerations of toxicity and ease of preparation
and storage, silver thiosulfate was used in subsequent experiments.
The method used for the preparation of silver thiosulfate was as
follows: 1.98 grams of sodium thiosulfate (pentahydrate) was
dissolved in 80 mL of water. 20 mL of a 0.1M solution of silver
nitrate was added while stirring vigorously, resulting in 100 mL of
a 20 mM stock solution of silver thiosulfate. Potassium thiosulfate
could be used in place of sodium thiosulfate with equally
efficacious results. The stock solutions were filter-sterilized
using 0.22 .mu.M cartridge filters into cell culture media at the
start of a given experiment. Alternative methods for preparing
similar silver thiosulfate solutions are also suitable. The cell
culture protocols were similar to those described for the
experiments described in Table 10.
[0178] Table 11 summarizes data obtained by using silver as an
enhancement agent for a number of different cell cultures of Taxus
chinensis. These data show that silver effects a fundamental
enhancement of taxane biosynthesis generally. The specific product
profile observed in any given case reflects characteristics of the
cell line and the culture medium. Silver ion/complex can be
particularly effective in enhancing taxane production when used in
conjunction with other factors in the medium favoring biosynthesis
such as growth regulators, carbon source, salts, micronutrients,
and the like.
Example 11
Enhancement of Taxane Production Using Methyl Jasmonate and
Jasmonate-Related Compounds
[0179] The methyl ester of jasmonic acid (methyl jasmonate), as
well as jasmonic acid and related compounds, were found to be
useful as enhancement agents of taxane biosynthesis in cell
cultures of Taxus species. The combination of methyl jasmonate and
other enhancement agents has also been found to be useful in
obtaining and sustaining high rates of taxane production.
[0180] Seven-day old cells of Taxus chinensis suspensions
cultivated in Medium M (Table 2) were suction filtered aseptically
using a sterile Buchner funnel fitted with a MIRACLOTH (Calbiochem)
filter. Cells were inoculated into culture medium of the given
composition indicated in Table 12, at a fresh weight cell density
in the range of 15% to 20% (w/v). The cultures were incubated at
24.+-.1.degree. C. at 120 or 180 RPM (depending on the vessel size)
on a gyratory shaker (1'' throw) in the dark. Evaporation was
corrected for by adding sterile distilled water. Samples of whole
broth (i.e., both extracellular and intracellular taxanes) were
taken at periodic intervals, and were processed and analyzed by
HPLC according to the methods outlined in Example 5.
[0181] Table 12 summarizes data obtained by using jasmonic acid and
its methyl ester as enhancement agents for several representative
Taxus chinensis cell lines. These data show that jasmonic acid and
its methyl ester effect a fundamental enhancement of taxane
biosynthesis generally. The specific product profile observed in
any given case reflects characteristics of the cell line and the
culture medium. These levels of production obtained in the presence
of these enhancing agents are significantly higher than that
observed in unenhanced cultures (the production levels for which
are elaborated in Example 7).
[0182] Jasmonic acid, its methyl ester, and related compounds, are
effective enhancement agents of taxane biosynthesis when used in
conjunction with other factors in the medium favoring biosynthesis
such as other enhancement agents, growth regulators, carbon source,
salts, micronutrients, and the like.
Example 12
Enhancement of Taxane Production Using
3,4-Methylenedioxy-6-nitrocinnamic acid
[0183] The cinnamic acid analog, 3,4-methylenedioxy-6-nitrocinnamic
acid (MDNA) and related compounds were found to be useful
enhancement agents of taxane biosynthesis in cell cultures of Taxus
species. The combination of MDNA and other enhancement agents has
also been found to be useful in obtaining and sustaining high rates
of taxane production.
[0184] Seven-day old cells of Taxus chinensis suspension culture
SS122-42 cultivated in Medium M (Table 2) were suction filtered
aseptically using a sterile Buchner funnel fitted with a MIRACLOTH
(Calbiochem) filter. Cells were inoculated into culture medium
conditions at a fresh weight density of 15% to 20% (w/v). The
vessels were incubated at 24.+-.1.degree. C. at 180 RPM on a
gyratory shaker (1'' throw) in the dark. Treated cultures were
sampled and analyzed using the methods described in Example 5 at
various time points. Evaporation was corrected for by adding
sterile distilled water at periodic intervals. Samples of whole
broth (i.e., both extracellular and intracellular taxanes) were
taken at periodic intervals, and were processed and analyzed by
HPLC according to the methods outlined in Example 5.
[0185] Table 13 summarizes data obtained by using
3,4-methylenedioxynitrocinnamic acid as an enhancement agent for
taxane biosynthesis in Taxus chinensis cell cultures. These data
show that MDNA effects a fundamental enhancement of taxane
biosynthesis generally. Cultivation in Medium II i.e., in the
presence of MDNA and silver, further enhances the production of
taxanes. The specific product profile observed in any given case
reflects characteristics of the cell line and the culture medium.
These levels of production are significantly higher than that
observed in unenhanced cultures (the production levels for which
are elaborated in Example 7).
Example 13
Enhancement of Taxane Biosynthesis Using a Combination of
Enhancement Agents
[0186] Various enhancement agents, used in combination, gave
significant and synergistic improvements in taxane production.
[0187] Seven-day old cells of Taxus chinensis suspension cultures
cultivated in Medium P (SS64-412), Medium O (SS64-561, SS64-571),
Medium I (SS124-77, SS85-26), Medium M (SS122-29) (the composition
of these media are listed in Table 2) were suction filtered
aseptically using a sterile Buchner funnel fitted with a MIRACLOTH
(Calbiochem) filter. Cells were inoculated into culture medium
(indicated in Table 14) at a fresh weight density of 20% (w/v). The
cultures were incubated at 24.+-.1.degree. C. at 180 RPM on a
gyratory shaker (1'' throw) in the dark. Evaporation was corrected
for by adding sterile distilled water at periodic intervals.
Samples of whole broth (i.e., both extracellular and intracellular
taxanes) were taken at periodic intervals, and were processed and
analyzed by HPLC according to the methods outlined in Example
5.
[0188] Table 14 summarizes data obtained by using various
combinations of enhancement agents for taxol, baccatin III, and
taxane biosynthesis in Taxus chinensis cell cultures. The data
demonstrates substantial further enhancement of taxane production
by combinations of enhancement agents over that seen for individual
agents, and over production levels in unenhanced conditions (the
production levels for which are elaborated in Example 7).
Example 14
Enhancement of Taxane Production by Medium Exchange
[0189] This example demonstrates that high productivity in culture
can be sustained by replenishing medium components and removing
spent medium.
[0190] Cell lines were initially cultivated in Medium O (Paella),
Medium I (SS29-3A5), and Medium I (SS45-146). The detailed
compositions of these cultivation media are described in Table 2.
Seven day-old cells of these cell lines were suction-filtered
aseptically using a sterile Buchner funnel fitted with a MIRACLOTH
(Calbiochem) filter. Approximately 1.5 grams fresh weight cells
were inoculated into 4.25 mL of the respective culture media
indicated in Table 15. The vessels were incubated at
24.+-.1.degree. C. at 120 RPM on a gyratory shaker (1'' throw) in
the dark. Evaporation was corrected for by addition of sterile
distilled water at periodic intervals. For the medium exchange
treatments, the spent production medium was suctioned off using a
sterile pipette after 10 to 11 days of batch cultivation, leaving
the cells behind in the vessel. The spent supernatant was analyzed
for extracellular taxanes using the methods described in Example 5.
Fresh culture medium of the same composition as the first batch
culture was added to the vessel containing productive cells. The
cells were cultured under the same environmental conditions
described above. The medium exchange cycle was repeated after an
additional 10 to 11 days of cultivation. The total extracellular
taxanes for batch production is compared with that of medium
exchange production in Table 15. The medium exchange concentration
values denote the total amount of taxane produced in the
extracellular medium divided by the volume of the cell suspension
culture (i.e., 5.75 mL).
[0191] Table 15 indicates that cells can be sustained in a
productive state for a prolonged period, and in fact, that
productivity of the cells can be enhanced by repeated medium
exchange. Enhancement by repeated medium exchange is feasible using
a range of different enhancement conditions, and with a variety of
cell cultures.
[0192] The data demonstrates substantial further enhancement of
taxane production over production levels in unenhanced conditions
(the production levels for which are elaborated in Example 7).
Example 15
Enhancement of Taxane Production by Fed Batch Operation
[0193] Seven day-old cells of cell lines cultivated in Medium I
(CR-128, SS36-245), Medium L (SS36-359) (the compositions of these
media are described in Table 2) were suction filtered aseptically
using a sterile Buchner funnel fitted with a MIRACLOTH (Calbiochem)
filter. Approximately 1 gram fresh weight of cells were inoculated
into 4 ml of culture medium of the given composition indicated in
Table 16.a. The vessels were incubated at 24.+-.1.degree. C. at 120
RPM on a gyratory shaker (1'' throw) in the dark. Evaporation was
corrected for by addition of sterile distilled water at periodic
intervals. For fed batch operation, sterile feed solutions of
predetermined compositions were fed continuously into the culture
vessels at predetermined rates of feeding, e.g. 10 mL feed solution
per liter of culture per day. Details of the fed batch operation
are described in Table 16.b., including compositions of the feed
solutions and feeding protocols. Treated cultures were sampled and
analyzed using the methods described in Example 5.
[0194] Table 16.a. indicates that cells can be sustained in a
productive state for a prolonged period, and in fact, that
productivity of the cells can be enhanced by fed batch operation,
resulting in the accumulation of high levels of baccatin III,
taxol, and other taxanes. The relative amounts of particular
taxanes reflect the interaction of feeding protocol and feed
composition with the cell line and culture conditions. This Table
also indicates that feeding phenylalanine results in enhanced
production of taxol relative to other taxanes.
[0195] The data demonstrates substantial further enhancement of
taxane production over production levels in unenhanced conditions
(the production levels for which are elaborated in Example 7).
Example 16
Enhancement of Taxane Biosynthesis Using a Combination of
Enhancement Agents
[0196] Various enhancement agents, used in combination, gave
significant and synergistic improvements in taxol, baccatin III,
and taxane production.
[0197] Seven-day old cells of Taxus chinensis suspension cultures
(SS122-41, cr427, SS122-30, cr857, cr452) cultivated in Medium M
(the composition of the medium is listed in Table 2) were suction
filtered aseptically using a sterile Buchner funnel fitted with a
MIRACLOTH (Calbiochem) filter. Cells were inoculated into culture
medium (indicated in Table 17) at a fresh weight density of 20%
(w/v) unless described otherwise in Table 17. The cultures were
incubated at 24.+-.1.degree. C. at 180 RPM on a gyratory shaker
(1'' throw) in the dark. Evaporation was corrected for by adding
sterile distilled water as necessary. Samples of whole broth (i.e.,
both extracellular and intracellular taxanes) were taken at
periodic intervals, and were processed and analyzed by HPLC
according to the methods outlined in Example 5.
[0198] Table 17 summarizes data obtained by using various
combinations of enhancement agents for taxol and taxane
biosynthesis in Taxus chinensis cell cultures. The data
demonstrates substantial further enhancement of taxane production
by combinations of enhancement agents over that seen for individual
agents, and over unenhanced conditions (the details of which are
provided in Example 7).
Example 17
Enhancement of Taxane Production by Fed Batch Operation
[0199] Seven day-old cells of cell lines cultivated in Medium M
(SS122-41) (the compositions of these media are described in Table
2) were suction filtered aseptically using a sterile Buchner funnel
fitted with a Miracloth (Calbiochem) filter. Approximately 1 gram
fresh weight of cells were inoculated into 4 ml of culture medium
of the given composition indicated in Table 18.a. The vessels were
incubated at 24.+-.2.degree. C. at 120 RPM on a gyratory shaker (1
throw) in the dark. Evaporation was corrected for by addition of
sterile distilled water. For fed batch operation, sterile feed
solutions of predetermined compositions were fed continuously into
the culture vessels. Details of the fed batch operation, including
compositions of the feed solutions and feeding protocols are
described in Table 18.b. Treated cultures were sampled and analyzed
using the methods described in Example 5.
[0200] Table 18.a. indicates that cells can be sustained in a
productive state for a prolonged period, and in fact, that
volumetric productivity of the cells can be enhanced by fed batch
operation, resulting in the accumulation of high levels of baccatin
III, taxol, and other taxanes. The relative amounts of particular
taxanes reflect the interaction of feeding protocol and feed
composition with the cell line and culture conditions.
[0201] The data demonstrates substantial further enhancement of
taxane production over production levels in unenhanced conditions
(the production levels for which are elaborated in Example 7).
[0202] For purposes of clarity of understanding, the foregoing
invention has been described in some detail by way of illustration
and example in conjunction with specific embodiments, although
other aspects, advantages and modifications will be apparent to
those skilled in the art to which the invention pertains. The
foregoing description and examples are intended to illustrate, but
not limit the scope of the invention. Modifications of the
above-described modes for carrying out the invention that are
apparent to persons skilled in the art are intended to be within
the scope of the invention, which is limited only by the appended
claims.
[0203] All publications and patent applications mentioned in this
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All publications and
patent applications are herein incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference.
TABLE-US-00008 TABLE 1.a List of Elicitors Used in Elicitation of
Taxus spp. Cell Cultures I. Biotic Elicitors (microorganisms)
Botrytis cinerea Phytophthora megasperma Pinellas stripticum
Oligosporus sp. Pythium mamillatum Pythium sylvaticum Verticillium
dahliae Verticillium sp. Penicillium minioluteum Phytophthora
lateralis Cytospora cincta Cytospora leucostoma Alternaria
brassicicola Alternaria solani Alternaria cucumerina Botrytis
squamosa Cochliobolus heterostrophus Colletotrichum trifolii
Colletotrichum orbiculare Colletotrichum graminicola Colletotrichum
gloeosporioides Cylindrocladium floridanum Fusarium crookwellense
Fusarium heterosporium Fusarium oxysporum f. sp. conglutinans
Fusarium oxysporum f. sp. lycopersici Fusarium oxysporum f. sp.
pisi Gibberella zeae Gaeumannomyces graminis var. tritici
Geotrichum sp. Leptosphaeria korrae Nectria haematococca MPVI
Mycosphaerella pinodes Ophiostoma ulmi Phoma lingam Phoma pinodella
Phytophthora infestans Pythium aristosporum Pythium graminicola
Pythium ultimum Rhizoctonia solani Sclerotinia sp. S. nodorum D-45
Trametes versicolor Ustilago maydis Venturia inaequalis II. Biotic
Elicitors (Microbial fractions or products) Chitosan Cellulysin
Lichenan Multifect XL Glucomannan Multifect CL Pleuran Resinase
Glucan Pulpxyme Carboxymethylglucan SP431 Hydroxymethylglucan
Pectinol Sulfoethylglucan Rapidase Mannan Klerzyme Xylan Chitinase
Mannobiose Mannotriose Mannopentaose Mannotetraose III. Abiotic
Elicitors (Chemical Stress Agents as well as some naturally
occurring biochemicals) Arachidonic acid Elaidic acid Cyclic AMP
Dibutyryl Cyclic AMP Methyl jasmonate Cis-Jasmone Miconazol Ferulic
acid AMO-1618 Triton X-100 Benzoic acid and derivatives Salicylic
acid and derivatives Propyl gallate Sesamol Chlorocholine chloride
3,4-dichlorophenoxy triethyl (amine) Chloroethylphosphonic acid
Diethyldithiocarbamic acid Nordihydroguaiaretic acid Sodium
metabisulfite Dithiothreitol b-amino-DL-Phenylalanine Potassium
metabisulfite Uniconazol Vanadyl sulfate Spermine Paclobutrazol
Putrescine Spermidine Cadavarine Protamine Sulfate SKF-7997 MER 29
Ancymidol Triadimefon Phosphon D Thiourea Dextran Sulfate
Hydroquinone Chitosan glutamate Fenpropemorph Prochloraz Naptifine
EDU HTA MPTA Glutathione EGTA Gibberellins Abscisic Acid
1,3-Diphenyl urea Diazolidinyl urea Phloroglucinol Sodium alginate
Carragenan
TABLE-US-00009 TABLE 1.b List of Precursors, Inhibitors &
Stimulants or Activators Used in Regulation of Biosynthesis of
Taxol & Taxanes in T. spp. cell cultures. Precursors Inhibitors
Stimulants Phenylalanine Chlorocholine chloride Cyclic AMP Lysine
Uniconazol Dibutyryl Cyclic AMP Tyrosine Paclobutrazol Methyl
jasmonate Tryptophan SKF-7997 Cis-Jasmone Methionine MER 29
Chloroethylphosphonic acid Tyramine Ancymidol Spermine Acetic acid
and Triadimefon Spermidine its' salts Phosphon D Putrescine
Mevalonic acid Fenpropemorph Cadavarine Farnesyl acetate Prochloraz
MPTA Geranyl acetate Naptifine DCPTA Geranylgeraniol Miconazol
DIPTA acetate Tryptamine Silver Nitrate ACC Menthol Norbornadiene
HTA a-Pinene AMO 1618 Brassinosteroids Trans-cinnamic acid Alar BHA
Cambrene A 4-amino-5-Hexynoic acid BHT Verticillene
Phenylethanolamine OTA Verticillol Phenethylamine Camphor
Glyphosate Quercetin Dihydrocycloeucalenol Levulinic acid
Methionine Sulfoxide Abietic acid .beta.-Hydroxyphenethylamine
Borneol 5-Methyl-DL-Tryptophan a-Fluorophenylalanine 5-2
Aminoethyl-L-cysteine hydrochloride
TABLE-US-00010 TABLE 1.c ELICITORS Xylanase Butaclore
Chitooligosaccharides Butylisothiocynate Spermine Bis Nitric oxide
Adduct Chloramben N,N'-Diacetylchitobiose isopropylamine Ethyl
carbamate Bis 2-Hydroxyethylhydrazine Nitric oxide Adduct
Hydroxyglutaric acid disodium Diethylamine Bis (Nitric oxide)
Adduct Tryptophol Benzyl N,N'-Diacetyl-.beta.-chitobioside Thiourea
Syringic acid Thioacetamide Benzothiadiazole 2,4,6-Trichlorphenol
Bipyridyl Pyridine-2-aldoxime methochloride Gossypol and
derivatives Potassium oxalate monohydrate
2-chlor-4-methylisonicotinic acid Poly-L-Lysine hydrobromide
Indomethacin Nerol N,N',N'-Triacetylchitotriose N-(1-Naphthyl)
phthalamic acid N,N'-Diacitylchitobiose Oxalate Diammoniun oxalate
Octapomine hydrochloride Nigeran Oxizamide p-hydroxyacetophenone
2-Methylpyrazine Pectic acid Methoxyacetic acid Lysozyme
N-Ethoxycarbonyl-2-ethoxy-1,2- Nitric oxide Dihydroquinoline
Glutathione (reduced) Lanthanum acitate 1,2-Diaminopropane
Linolenic acid 1,3-Diaminopropane Lipase .beta.-mercaptoethylamine
Iodoacetamide Hydroxylamine 2-hydroxyethylhydrazine Deoxyglucose
Dinocap 2-chlorobenzoic acid 1,3-Diphenylurea 2-Methyl-1,2-DL
(3-Pyridyl) 1-Propane Hydrogen peroxide 5-Bromouracil Urea
hydroperoxide 7-Nitrondazole Sebacic acid 8-Hydroxyquinoline
Benzoyl peroxide Acedoamidocinnamic acid N-methylmaleimide
2-Aminoanthraquinone Cumen peroxide N-Acetyl-L-glutamic acid
N-Acetyl-D-Glucosamine Agmatin Octyl-.beta.-D-Glucopyranoside
3-Acetyl pyridine Diisopropyl fluorophosphate Butyryl Butyryl
Lactate Isopropyl-.beta.-D- thiogalactopyranoside
7-Bromo-5-chloro-8-hydroxyquinoline Hydroxyethyl-.beta.-1,3-glucan
Benzylbenzoate Dextran Bromoxynil Lucifer yellow Syringaldehyde
Chitinase Bacitracin Calcium cyanide Glucans Glutaric acid
Morpholine Octamethylcyclotetrasiloxane Trigonelline hydrochloride
Anthranilic acid Colistin methane sulfonate Colchicine
2,4-Dichlorophenol L-Phenylalanine-2-naphthylamide Hydroxyglutaric
acid, and its salts DL-2-Hydroxy-3-methylbutyric acid
1-10-Phenanthroline monohydrate N-sulfosuccinimidyl-3-(4-
Hydroxyphenyl)propionate Trans-1,6-diphenylhexatriene Arachidonic
acid Urea hydrogen peroxide Hydrogen peroxide Bestatin Butylated
hydroxyanisole Butylated hydroxytoluene Gellan gum cellulase
Pimelic acid Diisopropyl phosphochloridate Nitrapyrin t-Butyl
hydroperoxide DL-Phosphinothricin ammonium Methyl syringate
Trifluralin Tridecanone Mimosine Narigenin Dimethylaminopyridine
1-Benzylimidazole DL-o-chlorophenylalanine Cetylpyridinium chloride
Hydroquinone Syringomycin
TABLE-US-00011 TABLE 1.d PRECURSORS Dimethylphenylalanine
D-fructose-1,6-Diphosphate Geranyl chloride .beta.-Hydroxypyruvic
acid Geranylgeraniol 4-Hydroxyphenylpyruvic acid trans-Cinnamic
acid Methyl acetate Pyruvic acid Methyl laurate Phenylpyruvic acid
Oxaloacetic acid Orthosuccinylbenzoic acid Pinenes
2,3-dihydrobenzoic acid Geranyl acetate o-hydroxyphenylpyruvic acid
Nerol Postassium acetate Phellandrene Glutamic acid Benzoyl
chloride Aspartic acid R(-)Citramalic acid DL-.beta.-phenylserine
Aspargine Hippuric acid 2,3-Dichlorobenzoic acid p-Hydroxycinnamic
acid Isoleucine Benzyl acetate Leucine Phenylacetic acid
Phosphoglyceric acid 3-Benzoylpropionic acid Serine Citric acid
2-Hydroxycinnamic acid Calcium benzoate 3-Hydroxycinnamic acid
Arginine 4-Hydroxycinnamic acid N-Benzoyl-DL-Phenylalanine Borneol
3,4-Dihydroxycinnamic acid Phosphoglycerate Potasium Salt
Phosphoenolpyruvic acid Glyceraldehyde-3-phosphate Phenylisoserine
Dihydroxyacetone phosphate 4-Hydrocoumarin Glycine Glutamine Ethyl
acetate Ornithine Methyl cinnamate Methionine Potassium acetate
Shikimic acid DL-Glyceraldehyde-Phosphate free Oxoglutamic acid
acid DL-3-Amino-3-phenylpropionic Calcium benzoate acid Oxoglutamic
acid a-Phenylalanine Phosphoenolpyruvic acid .beta.-Phenylalanine
Menthol N-Benzoylphenylisoserine Cambrene A Geraniol Verticillol
Linalool Verticellene Geranyl linalool Abietic acid Isoborynyl
isovalerate Succinic acid Cinnamyl acetate Fumaric acid Cinnamyl
propionate Acetoacetate Potasium Salt Cinnamyl chloride
TABLE-US-00012 TABLE 1.e INHIBITORS Rhizobitoxine
Trans-3,4-difluorocinnamic acid a-Canaline Mercaptoethanol
a-Aminosobutyric acid 4-Hydroxycoumarin cis-Propenylphosphonic acid
Cinnamulfluorene Flurprimidol 2-Cyano-4-Hydroxycinnamic acid
Chloromethyl Cyclopropane Cinnamylidenemalonic acid
Diazocyclopentadiene 4-Dimethylaminocinnamic acid Diammonium
succinate N-Cinnamylpiperazine g-Glutamylmethylamide
N-trans-Cinnamoylimidazole 2,3-Dimercaptosuccinic acid
Cinnamylideneacetophenone p-Nitrophenylphosphate 3,4-Methylenedioxy
cinnamic acid Pervanadate 3,4-Methylenedioxy-6-nitrocinnamic acid
Orthovanadate 3-(3,4-Methylenedioxyphenyl) propionic acid
N-Acetyl-DL-homocysteine Thiolactone 3,4-Methylenedioxyphenylacetic
acid 2,3-diphosphoglyceric acid salts 3,4-trans-Dimethoxycinnamic
acid p-Hydroxymercurylbenzoate 4-Methoxycinnamic acid Methylmercury
chloride 2-Methoxycinnamic acid Methylcyclopropane 4-Nitrocinnamic
acid ethyl ester Methylcyclopropane carboxylate Methoxycinnamic
acid Cyclooctodine 4-Nitrocinnamaldehyde Methoxyvinyl glycine
3-Nitrocinnamic acid Ibuprofen 2-Nitrocinnamic acid Piperonylic
acid 3,4-Dimethoxy-6-nitrocinnamic acid Phenylpropiolic acid
Ammonium oxalate L-2-Hydroxy-3-phenylpropionic acid Sinapic acid
Amino oxyacetic acid 2-Hydroxy-4,6-dimethoxybenzoic acid
D-Phenylalanine 3-dimethylaminobenzoic acid Phenylpyruvic acid
3,4-dimethoxybenzoic acid L-Tyrosine 4-Methoxybenzoic acid
4-Fluoro-(1-amino-2-phenylethyl) Phosphonic acid
N(G)-Nitro-D-Arginine 4-Hydroxyphenylpyruvic acid
N(G)-Nitro-L-Arginine m-Fluoro-DL-phenylalanine Malonic acid
p-Fluoro-DL-phenylalanine Maleic acid hydrozide
m-Fluoro-DL-tyrosine Okadaic acid 3,4-Difluoro-D-phenylalanine
1,4-Cyclohexanedione 1-Aminobenzotriazol Diisopropyl
fluorophosphate 4-Fluorocinnamic acid Oxamic acid SKF-525A Oxamic
acid, derivatives Diethyldithiocarbamic acid, Sodium Salt
Sulfanilamide Dithiothreitol N-Acetyl-S-farnesyl-L-cysteine
p-Coumaric acid Chaetomellic acid A, sodium salt Vinylimidazole
Isonicotinic acid hydrazide a-Hydroxyfarnesylphosphonic acid
2,3-dimercaptopropanol N6-Monomethyl-L-arginine Salicylhydroxyamic
acid 7-Nitroondazole 3-amino-4-hydroxybenzenesulphonic acid
Norflurazon Hydroxyurea Cyclooctodiene.alpha.-Fluorophenylalanine
6,7-dimethoxy-1,2-benzisoxazole-3-acetic acid Diethyldithiocarbamic
acid 3-oxo-1,2-benzisothiazoline-2-ylacetic acid
SKF-7997[Tris-(2-diethylaminoethyl)- 2,3,5-Triidobenzoic acid
phosphate trichloride] 2-(p-Chlorophenoxy)-2-methylpropionic acid
Triadimefon N-(1-Naphthyl)phthalamic acid 2,3,4-Trimethoxycinnamic
acid 1-Pyrenoxylbenzoic acid 2,4-Dimethoxycinnamic acid
2-Chloro-9-hydroxyfluorene-9-carboxylic acid 3-Hydroxyphenylacetic
acid Chlorocholine chloride 4-Aminotriazole
2'-Isopropyl-4'-(trimethylanmonium 4-Fluorocinnamic acid
chloride)-5-methyl phenylpeperidone carboxylate
4-Chloro-2-methylphenoxyacetic acid Sesamol 1,3-Dichloropropane
Ancymidol N-Ethylmaleimide Daminozide Semicarbizide Lovastatin
4-Chlororesorcinol Simvastatin 1,2-Dichloropropane Caffeic acid
Idoacetamide Ferulic acid Phenylhydrazine 2,5-Dihydroxycinnamic
acid Silver thiosulfate 2,5-Dihydromethoxycinnamic acid Silver
chloride 4-Hexylresorcinol Thiosemicarbazide Cetylpyridinum
chloride N-(phosponomethyl)-Glycine Stourosporine
p-Chlorophenoxyisobutyric acid Dimethylthiourea Triton x-100
Phenylpropiolic acid Triparanol Ammonium oxalate Chlorphonium
chloride 1-Aminobenzotriazole Mepiquat 1-Vinylimidazole
Prohexadione calcium salt Mercaptoethanol Chloromequat
3,5-Diido-4-hydroxybenzoic acid Tetcyclasis
5-Methyl-7-chloro-4-ethoxycarbanylmethoxy-
2-Aza-2,3-dihydrosqualene 2,1,3-benzothiadiazole Dinoconazole
Bromoxynil Tridemorph 3,4,5-Trichlorophenol 2,3-Iminosqualene
N-Methylmaleimide Glyphosine Ethyl-3-nitrocinnamate
Isoprophyl-N-phenyl carbamate 4-Fluoro-DL-tyrosine Oryzalin
Ethyl-3-nitrocinnamate Caffeine Conavanin D-Arginine
Methylacetylenic putrescine .alpha.-Methylornithine Methylpyruvic
acid Conavanine .alpha.-Hydroxy-2-pyridinemethane sulfonic acid
Abscisic acid Acetohydroxamic acid 3-Amino-1,2,4-triazole
Isopropyl-N-phenyl carbamate 4-Nitrocinnamic acid D1-phenylene
iodonium 3,4-Dimethoxyphenylacetic acid 2-Aminoindan-2-phosphonic
acid N-Cinnamylpiperazine Potassium-arsenate Hydroxylamine
.alpha.-aminooxy-.beta.-phenylpropionic acid
2,4-Dinitrophenylhydrazine Benzyl hydroxylamine Tetramethylammonium
bromide Piperonyl butoxide Clotrimazole Valinonycin Procaine
Monensin Uniconazole Paclobutrazole 4-Aminotriazole Benzyl
isothiocyanate Selenomethionine 1-Acetyl-2-thiourea
3,4-Dehydro-DL-proline 2-Ethylnaphthalene 3-Nitrobenzoic acid
Silver salts such as Silver chloride, Silver nitrate, etc. Sodium
hydrosulfite 7-nitronadozole Ethionine Azacytididine
Ethoxy-carbonyl-pyrimidine Miconazole
2,3:4,6-Di-o-isopropylidene-2-keto-L- Gulonic acid
N-(4-Hydroxyphenyl)glycine 3-(4-Hydroxyphenyl)propionic acid
3-(2-Hydroxyphenyl)propionic acid 4-Cyclohexanedione
N-(6-aminohexyl)-5-chloro-1-Naphthalene- sulfonamide hydrochloride
Endothal Phosphan Cyanamide .alpha.-(1-Methylethyl)-.alpha.-(4-
trifluoromethoxy)phenyl-5- pyrimidinemethanol 2-Aminoisobutyric
acid D-Arginine n-Butylamine p-Chloromercurybenzene sulphonic acid
Methylglyoxal bis (guanyl hydrazone) .alpha.-Methyl ornithine
TABLE-US-00013 TABLE 1.f STIMULANTS Potassium pyrophosphate
p-aminohippuric acid Sodium pyrophosphate Benzylcinnamate Uracil
Jasmonic acid Melatonin Methyl jasmonate Hydroxylamine
hydrochloride Dihydroisojasmone Thionicotinamide Isojasmone
S-adenosyl-L-methionine cis-jasmone Inosine triphosphate
Tetrahydrojasmone Indole-3-lactic acid Lactone of cis-jasmone
Indole-3-pyruvic acid Dihydrojasmone Indole-2-carboxylic acid
Jasminolactone Indole-3-aldehyde Jasmolactone N-indolyl acetyl
valine 12-oxophytodienoic acid Pyridoxal phosphate Jasmonol Methyl
dihydrojasmonate g-methyldecalactone Bipyridyl Citronellyl tiglate
4-acetamidophenol Jasmonyl acetate Imidazole Mastoparan
Octyl-.beta.-D-glucopyranoside Lysophosphatidic acid
3-aminopyridine Cypermethrin Guanylic acid Cantharidin Citydylic
acid Acetylsalicylic acid Isopropyl-.beta.-d-thiogalactopyranoside
Salicylic acid and derivatives 3-(4-hydroxyphenyl) propionic acid
2,6-dichloroisonicotinic acid 3-(2-hyroxyphenyl) propionic acid
Nitric oxide Indole-3-pyruvic acid Traumatic acid Thiobenzoic acid
Citric acid Dimethylaminophenylalanine Cytidylic acid
p-hydroxyphenylpyruvic acid malic acid or malic acid salt
2,3-dihydroxybenzoic acid Potassium malate Ethyl benzoate Citric
acid salts and derivatives 3,4-dihydroxycinnamic acid Flavin
adenine mononucleotide 4-hydroxycinnamic acid Flavin monocleotide
N-acetyl-L-phenylalanine dibutyrl Cyclic AMP 3-Benzoylpropionic
acid Spermine p-hydroxycinnamic acid Spermidine 5',5'-Dithiobis
(2-nitrobenzoic acid) Putrescine .beta.-hydroxypyruvic acid
Cadavarine 4-hydroxyphenylpyruvic acid S-Adenosylmethionine Methyl
cinnamate Pyridoxal phosphate Methyl salicylate 6-Aminonicotinamide
2-napthylbenzoate 4-Dimethylaminopyridine Phenylsalicylate
N-(2-Hydroxyethyl)succinimide Thiosalicylic acid 2-oxoglutaric acid
Propachlor Thiamine Vinyl propionate Triethylamine hydrochloride
3,5-Diisopropylsalicylic acid Adenine sulfate
p-Amino-L-Phenylalanine Benzyl salicylate 1,2-Benzisoxazole
2,4-Carbonyldibenzoic acid L-Citrulline D-Erythrose 4-Phosphate
Fructose 1,6-Diphosphate Inosine triphosphate N-Methylputrescine
dihydrochloride .beta.-Phenylethylamine hydrochloride Lysine
Imidazole Guanylic acid Melatonin Aminocyclopropane-carboxylic acid
Isopentylpyrophosphate N-Acetyl-L-glutamine Isoglutamine Threonine
Potassium Pyrophosphate Sodium pyrophosphate L-2-Aminoadipic acid
N-methyl-N-Propagylbenzylamine hydrochloride Aminoguanidine
hemisulfate L-(+)-2-Amino-7-Phosphonoheptanoic acid Ammonium
sulfamate Spermine Bis Nitric oxide adduct Diethylamine Bis Nitric
oxide adduct Galactose Valine Vitamin B-12 Ascorbic acid and
derivatives Coronatine Phenobarbital Pregnenolone
24-epi-Brassinolide n-Propyl Dihydrojasmonate Propyl jasmonate
Epimethyl jasmonate
TABLE-US-00014 TABLE 2 Composition of media used for cultivation of
species cultures. Medium A B C D E F G H I J K L M N O P Chemical
Ingredient mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L
mg/L mg/L mg/L mg/L mg/L Ammonium Nitrate 400.0 500.0 400.0
Ammonium Sulfate 134.0 33.5 134.0 67.0 134.0 134.0 134.0 134.0
134.0 134.0 33.50 134.0 134.0 Boric Acid 3.0 1.5 0.75 3.0 1.5 0.75
6.2 1.5 3.0 3.0 3.0 3.0 3.0 0.75 3.0 3.0 Calcium Chloride 113.24
28.31 113.24 56.62 72.5 113.24 72.5 113.24 113.24 113.24 113.24
113.24 28.31 113.24 113.24 (anhydrous) Calcium Chloride 20.0 50.0
50.0 2-H2O Calcium Nitrate 208.4 386.0 386.0 4-H2O Cobalt Chloride
0.025 0.006 0.025 0.0125 0.025 0.025 0.025 0.025 0.025 0.025 0.01
0.025 0.025 6-H2O Cupric Chloride H2O 0.01 Cupric Sulfate 5-H2O
0.025 0.01 0.006 0.025 0.0125 0.25 0.025 0.25 0.025 0.025 0.025
0.025 0.025 0.01 0.025 0.025 Na2 EDTA 2-H2O 37.3 9.32 37.3 18.65
37.3 37.3 37.3 37.3 37.3 37.3 37.3 37.3 9.33 37.3 37.3 Ferric
Sulfate 2.5 Ferrous Sulfate 7-H2O 27.85 6.95 27.85 13.9 27.85 27.85
27.85 27.85 27.85 27.85 27.85 27.85 6.96 27.85 27.85 Magnesium
Sulfate 122.09 366.2 30.5 122.09 61.04 180.7 122.09 180.7 122.09
122.09 122.09 122.09 122.09 30.52 122.09 122.09 (anhydrous)
Manganese Sulfate 10.0 23.788 22.5 10.0 5.0 22.3 10.0 22.3 10.0
10.0 10.0 10.0 10.0 27.50 10.0 10.0 H2O Molybdenum Trioxide 0.001
Molybdic Acid 0.25 0.062 0.25 0.125 0.25 0.25 0.25 0.25 0.25 0.25
0.25 0.25 0.06 0.25 0.25 (sodium salt) 2-H2O Potassium Chloride
65.0 Potassium Iodide 0.75 0.75 0.175 0.75 0.375 0.75 0.75 0.75
0.75 0.75 0.75 0.19 0.75 0.75 Potassium Nitrate 2500.0 80.0 625.0
2500.0 1250.0 2500.0 2500.0 2500.0 2500.0 2500.0 2500.0 625.00
2500.0 2500.0 Potassium Phosphate 10.0 170.0 170.0 (monobasic)
Potassium Sulfate 990.0 990.0 Sodium Phosphate 130.5 16.5 32.62
130.5 65.25 130.5 130.5 130.5 130.5 130.5 130.5 32.63 130.5 130.5
(monobasic anhydrous) Sodium Sulfate 200.0 Zinc Sulfate 7-H2O 2.0
3.0 0.5 2.0 1.0 8.6 2.0 8.6 2.0 2.0 2.0 2.0 2.0 0.50 2.0 2.0
Myo-inositol 100.0 100.0 125.0 100.0 50.0 100.0 100.0 100.0 100.0
100.0 100.0 100.0 100.0 25.00 100.0 100.0 Nicotinic Acid 1.0 0.75
1.0 0.5 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.25 1.0 1.0 Pyridoxine HCL
1.0 0.25 1.0 0.5 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.25 1.0 1.0
Thiamine HCL 10.0 *5.0 3.5 10.0 5.0 10.0 10.0 10.0 10.0 10.0 10.0
10.0 10.0 2.50 10.0 10.0 Glutamine 292.8 146.4 292.8 292.8 1756.8
292.8 292.8 292.8 292.8 292.8 292.8 Tryptophan Phenylalanine 30.0
Lysine 20.0 Methionine Sodium Acetate 10.0 10.0 ucrose 10000.0
50000.0 40000.0 10000.0 10000.0 10000.0 20000.0 10000.0 10000.0
10000.0 10000.0 10000.0 50000.0 10000.0 10000.0 6 Benzyladenine
0.002 2.0 2.0 0.002 0.002 0.002 0.002 0.02 0.02 0.002 0.02
-Naphthaleneacetic 0.931 10.0 1.862 0.931 0.931 1.862 1.862 0.931
1.862 Acid Ascorbic Acid 50.0 100.0 50.0 100.0 100.0 100.0 100.0
100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 oram 1.2 2.4 1.2
1.2 2.4 asein Hydrolysate 500.0 1000.0 -Dimethylallyl- 0.02 amino)
Purine inetin 0.02 hidiazuron 0.022 altose 10000.0 Glutamic Acid
1850.0 1850.0 1850.0 1850.0 Aspartic Acid 1710.0 Glycine 5.0 Serine
5.0 Folic Acid 1.0 odium pH 5.6 5.8 5.8 5.6 5.6 5.6 5.6 5.6 5.6 5.6
5.6 5.6 5.8 5.6 5.6 Indicates that the indicates data missing or
illegible when filed
TABLE-US-00015 TABLE 3 Preferred conditions for callus
proliferation for various Taxus species. The ingredients in the
basal media are listed in Table 2. Basal Growth Regulators* Medium
Auxin Cytokinin Species (Table 2) Type Conc (M) Type Conc(M) T.
brevifolia F P 5 .times. 10-.sup.6 2iP 10-.sup.7 D P 5 .times.
10-.sup.6 BA 10-.sup.8 T. canadensis H P 5 .times. 10-.sup.6 K
10-.sup.7 D P 5 .times. 10-.sup.6 BA 10-.sup.8 T. chinensis D P 5
.times. 10-.sup.6 BA 10-.sup.8 A N 5 .times. 10-.sup.6 BA 10-.sup.8
T. globosa D P 5 .times. 10-.sup.6 BA 10-.sup.8 T. floridana D P 5
.times. 10-.sup.6 BA 10-.sup.8 T. baccata D P 5 .times. 10-.sup.6
BA 10-.sup.8 T. cuspidata D P 5 .times. 10-.sup.6 BA 10-.sup.8 T.
media D P 5 .times. 10-.sup.6 BA 10-.sup.8 T. wallichiana D P 5
.times. 10-.sup.6 BA 10-.sup.8 *Abbreviations: Picloram (P),
Naphthalene acetic acid (N), Benzyladenine (BA), Dimethyl
allylamino purine (2iP), Kinetin (K)
TABLE-US-00016 TABLE 4 Typical growth characteristics of Taxus sp.
suspension cultures Density Dry Weight Fresh Weight Fresh Doubling
Doubling Dry Wt. Species Time Time Density Wt. T. brevifolia g/L
2.0 days 3.5 days 20 g/L 400 T. baccata 2.0 6.0 15 220 T. chinensis
2.5 4.5 20 285 T. canadensis nd* 8.5 13 260 *not yet determined
TABLE-US-00017 TABLE 5 Taxol production in various Taxus species.
Taxol content Medium Species (% dry weight) (See Tables 2&3)
Analysis T. brevifolia 0.006 F ELISA T. canadensis 0.004 H ELISA T.
baccata 0.0014 D HPLC T. globosa 0.0003 G ELISA T. cuspidata 0.0025
G HPLC T. floridana 0.001 G ELISA T. media 0.02 F ELISA T.
chinensis 0.18 B HPLC
TABLE-US-00018 TABLE 6 Improvements in productivity due to medium
exchange treatment. Numbers are expressed as X-fold improvement
over levels achieved in a 15-day batch interval. Taxus chinensis
cell line K-1 was cultivated in Medium A in the dark. Total levels*
Extracellular levels Taxol 4.6 4.89 Total taxanes 4.55 5.94 *Total
levels in cells and medium combined
TABLE-US-00019 TABLE 7 Effect of Standard GroLux light treatment on
taxol and taxane content in 10-day old cultures of Taxus chinensis
line K-1 cultivated in Medium A. Amounts shown are expressed as mg
extracted from 20 ml of suspension. Cell growth was identical in
both treatments (164 mg dry weight per flask). Light Dark Total
taxol: cells and medium: 8.8 .mu.g 3.13 .mu.g Extracellular taxol:
76.40% 56.20% Total taxanes cells and medium: 61.55 .mu.g 62.17
.mu.g Extracellular taxanes: 89% 84%
TABLE-US-00020 TABLE 8 Comparison of chitosan-glutamate treated to
non-elicited suspensions of Taxus chinensis line K-1 after 15 days
cultivation in medium C. Taxane levels reported are from cells and
medium combined. % extra refers to the percentage of extracellular
CONTROL ELICITOR Cell density 10.1 g/L Cell density 14.2 gm/l Cell
viability 70-80% viable Cell viability 75-80% viable % % % %
Taxanes dry wt mg/L Extra dry wt mg/L Extra Taxol 0.054 5.4 7.2
0.098 13.9 85.0 Baccatin III 0.057 5.8 69.9 0.055 7.8 76.6
7-Xylosyl-10- 0.040 4.0 63.0 0.048 6.9 77.0 deacetyltaxol
10-deacetyltaxol 0.0004 0.4 71.1 0.0 1.0 75.3 Cephalomannine
10-deacetylbaccatin III 10-deacetyl-7-epitaxol 0.054 5.4 74.2 0.076
10.8 85.7 7-Epitaxol 0.009 0.9 74.6 0.009 1.3 86.2 Unknown Taxanes
0.203 20.5 79.7 0.240 34.1 90.2 Total Taxanes: 0.421 42.4 0.533
75.8
TABLE-US-00021 TABLE 9 Nutrient medium manipulation for enhanced
taxane and taxol biosynthesis in Taxus chinensis suspension line
K-1. 500 mg fresh weight cells were inoculated per 5 mL of medium
and incubated in the dark for 18 days. The total taxanes produced
(in the cells and medium combined) is reported. The ingredients in
media B & C are listed in Table 2. Medium B Medium C Taxane
Level (mg/L) (mg/L) Baccatin III 4.3 3.9 7-xylosyl 10-deacetyl
taxol 8.3 12.9 Cephalomannine 1.1 trace 10-deacetyl 7-epi taxol 4.6
5.4 taxol 24.1 21.3 7-epi taxol 1.3 2.8 other unidentified taxanes*
56.1 63.7 Total taxanes 99.8 mg/l 110 mg/l
TABLE-US-00022 TABLE 10 Enhancement of Taxane Biosynthesis in Taxus
chinensis cell line KS1A by Silver Dose mg/L extracellular
product** Silver Compound (mM) Baccatin III Taxol Total Taxanes
Culture Medium only* 16 5 21 Silver thiosulfate 50 71 15 86 Silver
phosphate 100 48 7 55 Silver benzoate 20 40 7 47 Silver sulfate 20
61 7 68 Toluenesulfonic acid 20 39 6 45 silver salt Silver chloride
10 22 18 40 Silver oxide 50 43 18 61 Silver acetate 10 52 10 62
Silver nitrate 20 63 6 69 *The culture medium was Medium N from
Table 2, with the addition of the following growth regulators: 10
mM a-naphthaleneacetic acid, and 1 mM thidiazuron **All samples
were taken after 14 days of incubation.
TABLE-US-00023 TABLE 11 Enhancement of Taxol and Taxane
Biosynthesis by Silver in several Taxus chinensis cell lines. The
titers represent levels measured in the whole broth, i.e., in the
cells and in the extracellular medium. Other Total Cell
Silver.sup.a Culture Duration Baccatin III Taxol Taxanes Taxanes
Culture Concentration Medium (days) mg/L mg/L mg/L (mg/L) SS6A-1224
0 I.sup.b 30 10 48 23 81 SS6A-1224 50 mM I 30 172 86 126 384
SS122-13 0 II.sup.c 14 2 21 10 33 SS122-13 50 mM II 14 12 103 60
173 SS122-42 0 II 14 3 80 26 109 SS122-42 50 mM II 14 4 146 38 188
.sup.aAdded as silver thiosulfate .sup.bThe culture medium is
Medium N from Table 2, with the addition of the growth regulator,
a-naphthaleneacetic acid at a concentration of 10 mM. .sup.cThe
culture medium is Medium N from Table 2, with the addition of the
growth regulator, a-naphthaleneacetic acid at a concentration of 10
mM and thidiazuron at a concentration of 1 mM
TABLE-US-00024 TABLE 12 Enhancement of Taxol and Taxane
Biosynthesis by Jasmonic acid and its methyl ester. Taxane titers
were measured in the whole broth after 14 days of cultivation. The
culture medium was Medium N from Table 2, with the additional
presence of the growth regulator, a-naphthaleneacetic acid at a
concentration of 10 mM. Baccatin Other Total Cell Jasmonate III
Taxol Taxanes Taxanes Culture Concentration mg/L mg/L mg/L (mg/L)
SS122-42 0 3 80 26 109 SS122-42 200 mM JMA 4 120 87 211 SS122-42 89
mM MJS 3 121 109 233 SS122-13 0 2 21 10 33 SS122-13 89 mM MJS 9 73
63 124 *JMA denotes the free acid, and MJS denotes methyl
jasmonate
TABLE-US-00025 TABLE 13 Enhancement of Taxol and Taxane
Biosynthesis by 3,4-methylenedioxy- nitrocinnamic acid (MDNA).
Taxane levels were measured in the whole broth after 14 days of
cultivation. The cell line used was Taxus chinensis SS122-42. Other
Total MDNA Culture Baccatin III Taxol Taxanes Taxanes Concentration
Medium.sup.a mg/L mg/L mg/L (mg/L) 0 I 3 80 26 109 50 mM I 5 163 45
213 50 mM II 34 311 89 434 .sup.aThe culture medium I refers to
Medium N from Table 2, with the additional presence of the growth
regulator, a-naphthaleneacetic acid at a concentration of 10 mM.
The culture medium II is identical to Culture medium I, with the
additional presence of 50 mM silver thiosulfate.
TABLE-US-00026 TABLE 14 Enhancement of Taxol and taxanes in cell
cultures of Taxus chinensis using various combinations of
enhancement agents. All taxane concentrations are expressed as
whole broth titers (i.e., concentration in cells and medium
combined), and values were obtained after 11 days of incubation.
Other Total Cell Culture Baccatin Taxol Taxanes Taxanes Culture
Medium.sup.a mg/L mg/L mg/L (mg/L) SS64-412 I 41 464 101 606
SS64-561 II 590 182 388 1160 SS64-571 III 596 158 261 1015 SS124-77
IV 72 39 576 687 SS122-29 V 18 306 152 476 SS85-26 VI 586 100 416
1102 .sup.aThe culture medium for all combinations was Medium N in
Table 2. Culture Medium I contained, in addition to Medium N, 10 mM
a-naphthaleneacetic acid (NAA), 3 mM thidiazuron (TDZ), 50 mM
3,4-methylenediox-6-ynitrocinnamic acid (MDNA), 89 mM methyl
jasmonate (MJS), and 50 mM silver thiosulfate (SLTS). Culture
Medium II contained, in addition Medium N, 10 mM NAA, 1 mM TDZ, 50
mM MDNA, 89 mM MJS, 10 mM SLTS, and an additional 98.5 mg/L sodium
phosphate (monobasic). Culture medium III contained, in addition to
Medium N, 10 mM indolebutyric acid, 3 .mu.M TDZ, 30 mM
3,4-methylenediox-6-ycinnamic acid, 89 mM MJS, and 50 mM SLTS.
Culture medium IV contained, in addition to Medium N, 10 mM NAA, 89
mM MJS, 100 mM SLTS, and 5 mM glutamine. Culture medium V
contained, in addition to Medium N, 10 mM NAA, 89 mM MJS, and 50 mM
SLTS. Culture medium VI contained, in addition to Medium N, 10 mM
NAA, 1 mM TDZ, 50 mM MDNA, 18 mM MJS, 50 mM SLTS, and 5 mM
glutamine.
TABLE-US-00027 TABLE 15 Enhancement of Taxane Production by Medium
Exchange. Ave. Volumetric Culture Type of Duration Production
Productivity.sup.e Cell Line Medium.sup.a Operation.sup.b (days)
Product.sup.c Level.sup.d (mg/L) (mg/L/day) Paella I Batch 11 Taxol
185 13 Paella I Medium exchange 20 Taxol 265 17 SS29-3A5 II Batch
14 Baccatin III 260 18 SS29-3A5 II Medium exchange 28 Baccatin III
580 21 SS29-3A5 II Batch 22 10-deacetyl- 300 14 baccatin III
SS29-3A5 II Medium exchange 28 10-deacetyl- 400 14 baccatin III
SS45-146 III Batch 11 Total Taxanes 700 64 SS45-146 III Medium
exchange 28 Total Taxanes 2500 89 .sup.aThe culture medium for
these culture conditions was Medium N in Table 2. Culture medium I
included, in addition to Medium N, 10 mM a-naphthaleneacetic acid
(NAA), 1 mM thidiazuron (TDZ), 50 mM
3,4-methylenedioxynitro-cinnamic acid (MDNA), 18 mM methyl
jasmonate (MJS), and 10 mM silver thiosulfate (SLTS). Culture
medium II included, in addition to Medium N, 10 mM NAA, 1 mM TDZ,
50 mM MDNA, 89 mM MJS, 10 mM SLTS, and 5 mM glutamic acid
(monopotassium salt). Culture medium III included, in addition to
Medium N, 10 mM NAA, 2.5 mM zeatin, 30 mM MDNA, 89 mM MJS, and 50
mM SLTS. .sup.bRepeated enhancement was achieved by medium
exchange, as described in Example 14. .sup.cThe predominant product
produced by a given cell line under the specified culture medium is
listed; taxanes other than the predominant product were also
produced in each case, except for cell line SS45-146, for which
total taxane production is listed. .sup.dThe production levels for
batch cultivation refer to extracellular concentrations, i.e., the
amount of taxane measured in the extracellular medium divided by
the volume of the extracellular medium. For repeated enhancement by
medium exchange, the production level refers to the total amount of
taxane measured in the extracelluar medium after each medium
exchange, divided by the suspension volume. .sup.eThe average
volumetric productivity is one indicator of biosynthetic
capability; it is defined as the total product divided by the
suspension volume, and futher divided by the duration of the
incubation.
TABLE-US-00028 TABLE 16.a Enhancement of Taxol and Taxane
Production by Fed Batch Operation Total culture Baccatin Other
Total Culture Type of Fed batch duration III Taxol taxanes taxanes
Cell line medium.sup.a operation components.sup.b (days) (mg/L)*
(mg/L) (mg/L) (mg/L) CR-128 A Batch -- 24 152 134 203 489 A Fed
batch F1 24 257 200 295 752 A Fed batch F2 24 254 316 427 997
SS36-245 B Batch -- 31 170 80 190 440 B Fed batch F3 31 50 212 198
460 B Fed batch F4 31 56 412 348 816 SS36-359 C Batch -- 21 220 155
163 538 C Fed batch F5 21 439 182 304 925 .sup.aThe culture medium
for all cell lines was Medium N (Table 2). In addition, Culture
medium I contained 10 .mu.M .alpha.-naphthaleneacetic acid (NAA),
30 .mu.M 3,4-methylenedioxy-6-nitrocinnamic acid (MDNA), 18 .mu.M
methyl jasmonate (MJS), and 50 .mu.M silver thiosulfate (SLTS).
Culture medium II contained, in addition to Medium N, 10 .mu.M NAA,
50 .mu.M MDNA, 50 .mu.M SLTS, and 1 .mu.M thidiazuron (TDZ).
Culture medium III contained, in addition to Medium N, 10 .mu.M
NAA, 1 .mu.M TDZ, 50 .mu.M MDNA, 50 .mu.M SLTS, 89 .mu.M MJS. *All
taxane values refer to whole broth titers: (mg taxanes in cells +
mg taxanes in extracellular medium)/Total culture
volume(liters).
TABLE-US-00029 TABLE 16.b Details of fed-batch operation described
in Table 16.a. Duration Feed Feed rate Start of of feed solution
Composition (mL/L/day) feed (day) (days) F1 25% (weight/volume)
(w/v) fructose, 25 mM 10 7 17 gluamine, 50 .mu.M NAA, 250 .mu.M
SLTS, 89 .mu.M MJS, 1.48 mM calcium chloride, 0.63 mM magnesium
sulfate 0.68 mM sodium phosphite (monobasic) F2 F1, 75 mM
.alpha.-phenylalanine, 10 7 17 25 mM .beta.-phenylalanine F3 25%
(w/v) fructose, 10 6 25 150 mM .alpha.-phenylalanine, 25 mM
.beta.-phenylalanine F4 50% (w/v) glucose, 5.92 mM calcium
chloride, 2.52 5 9 22 mM magnesium sulfate, 2.72 mM sodium
phosphate (monobasic), 500 .mu.M SLTS, 10 .mu.M TDZ, 100 .mu.M NAA,
150 mM .alpha.-phenylalanine, 50 mM .beta.-phenylalanine F5
contained 50% (w/v) glucose, 5 12 9 100 .mu.M NAA, 10 .mu.M TDZ,
500 .mu.M SLTS, 89 .mu.M MJS, 0.68 mM sodium phosphate (monobasic),
50 mM .alpha.-phenylalanine
TABLE-US-00030 TABLE 17 Enhancement of Taxol and taxanes in cell
cultures of Taxus chinensis using various combinations of
enhancement agents. All taxane concentrations are expressed as
whole broth titers (i.e., concentration in cells and medium
combined). Other Total Cell Culture Duration Baccatin Taxol Taxanes
Taxanes Culture Medium.sup.a (days) mg/L mg/L mg/L (mg/L) SS122-41
I 20 106 374 158 638 SS122-41 I.sup.b 20 7 507 148 662 SS122-30 II
14 27 279 226 532 cr427 III 14 13 302 125 440 cr452 IV 14 11 190 95
296 cr452 V 14 4 172 67 243 cr857 I 24 116 531 258 905 cr914 VI 14
260 436 312 1008 .sup.aThe culture medium for all combinations was
Medium N (Table 2) in which the primary carbon source was replaced
by other sources as described in this legend. Culture Medium I
contained 100 g/l maltose instead of sucrose, and in addition,
contained, 20 mM 1-naphthaleneacetic acid (NAA), 40 mM
3,4-methylenedioxynitrocinnamic acid (MDNA), 45 mM methyl jasmonate
(MJS), 100 mM silver thiosulfate (SLTS), and 5 mM glutamine.
Culture Medium II contained 50 g/l maltose instead of sucrose, and
in addition, contained, 10 mM NAA, 40 mM MDNA, 100 mM MJS and 75 mM
SLTS. Culture medium III contained 50 g/L maltose instead of
sucrose, and in addition, contained, 20 mM NAA, 40 mM MDNA, 45 mM
MJS, 100 mM SLTS, and 5 mM glutamine. Culture medium IV contained
50 g/l lactose instead of sucrose, and in addition, contained, 20
mM NAA, 40 mM MDNA, 45 mM MJS, 100 mM SLTS, and 5 mM glutamine.
Culture medium V contained 40 g/l galactose instead of sucrose, and
in addition, contained, 20 mM NAA, 40 mM MDNA, 45 mM MJS, 100 mM
SLTS, and 5 mM glutamine. Culture medium VI contained 70 g/L
maltose instead of sucrose and in addition, contained, 20 mM NAA,
40 mM MDNA, 45 mM MJS, 100 mM SLTS, and 5 mM glutamine. .sup.bThe
fresh weight density was 26% (w/v).
TABLE-US-00031 TABLE 18.a Enhancement of Taxol and Taxane
Production by Fed Batch Operation Baccatin Other Total Cell Culture
Type of Fed batch III Taxol taxanes taxanes culture medium.sup.c
operation components.sup.d (mg/L).sup.e (mg/L) (mg/L) (mg/L)
SS122-41.sup.a A Batch -- 120 225 123 468 A Fed batch F1 32 476 171
679 A Fed batch F2 27 501 180 708 SS122-41.sup.b B Batch -- 7 507
148 662 B Fed batch F3 66 902 251 1219 .sup.aInoculation density
was 20% (w/v) .sup.bInoculation density was 26% (w/v) .sup.cThe
culture medium for all cell lines was Medium N (Table 2). The
primary carbon source was sucrose unless substituted as described
here. In addition, culture medium A contained 20 .mu.M
.alpha.-naphthaleneacetic acid (NAA), 40 .mu.M
3,4-methylenedioxynitrocinnamic acid (MDNA), 45 .mu.M methyl
jasmonate (MJS), and 100 .mu.M silver thiosulfate (SLTS), and 5 mM
glutamine. Culture medium B contained 100 mg/l maltose instead of
sucrose, and in addition contained, 20 .mu.M NAA, 40 .mu.M MDNA, 45
.mu.M MJS, 100 .mu.M SLTS, and 5 mM glutamine. .sup.dRefer to Table
18b .sup.eAll taxane values refer to whole broth titers: (mg
taxanes in cells + mg taxanes in extracellular medium)/Total
culture volume(liters)
TABLE-US-00032 TABLE 18.b Details of fed-batch operation described
in Table 18.a. Duration Start of fed Feed Feed rate of feed batch
solution Composition (mL/L/day) (day) (days) F1 50% (weight/volume)
8 10 11-21 (w/v) fructose, 50 mM glutamine F2 50% (w/v) maltose, 8
10 11-21 50 mM glutamine F3 50% (w/v) maltose, 8 10 10-20 200 .mu.M
NAA, 450 .mu.M MJS, 50 mM glutamine
* * * * *