Biotransformation in plant cells

1. BIOTRANSFORMATION OF
PHYTOCHEMICALS

2. Secondary Metabolites - Phytochemicals
• Plants represent and unlimited source of phyto-
chemicals such as the metabolites of primary
and secondary origin.
• The secondary compounds which are
biosynthetically derived from the primary
metabolites are of measure interest because of
their different functions and biological activities.
• They are sometimes considered to be waste or
secretor products of plant metabolism.
• Examples of secondary metabolites found in
plants glycosides, alkaloids, flavono

3. Biotransformation
• Biotransformation is an area of biotechnology that has gained
considerable attention.
• It is the ability of plant cell culture to catalyze the conversion of
readily available inexpensive precursor into a more valuable final
product.
• This precursor which cannot be transformed effectively by chemical
or microbial methods are an interesting area for commercial
application of plant tissue culture. This is a process of chemical
conversion of an exogenously supplied substance by living cell
culture.
• Plant-cells can transform a wide range of substrate and; thus perform
several reactions such as, oxidation, hydroxylation, reduction,
methylation, amino-acylation, glucosylation-acylation.
Biotransformation is the resent technique for commercial
exploitation of secondary metabolites from cell-culture. It can also be
defined as-chemical transformation which is catalyzed by micro-
organism or their enzymes.
• Enzymatically catalyzed biotransformation is superior to chemically
catalyzed reactions. The production of digitoxin from digitoxigenin by
Digitalis lanata culture is classical examples of utilizing plant cell
culture for achieving a specific biotransformation on a large scale.

4. Biotransformation
• Biotransformation means chemical alteration of chemicals
such as nutrients, amino acids, toxins, and drugs in the body.
• It is also needed to render nonpolar compounds polar so that
they are not reabsorbed in renal tubules and are excreted.
• Biotransformation of xenobiotics can dominate toxic kinetics
and the metabolites may reach higher concentrations in
organisms than their parent compounds.
• The conversion of molecules from one form to another within
an organism often associated with change (increase, decrease,
or little change) in pharmacologic activity.

5. Advantage of Biotransformation
• More than one reaction can be accomplished using
cell cultures that express a series of enzyme activities.
• In some instances even non producing cell culture
can be used to synthesize the desired end product
using appropriate precursor.
• The process of biotransformation may be simple
where the process is mediated by one or more
enzymes with many steps.
• Single step biotransformation is comparatively
efficient, as the yield decreases with increase in steps.
• Natural or synthetic substrate is used for
biotransformation.

6. • Useful compounds can be produced under
controlled conditions independent of climatic
changes or soil conditions.
• Cultured cells would be free of microbes and insects.
• The cells of any plants, tropical or alpine, could
easily be multiplied to yield their specific
metabolites.
• Automated control of cell growth and rational
regulation of metabolite processes would reduce
labor costs and improve productivity.
• Organic substances are extractable from callus
cultures.

7. Biotransformation and biotechnology
• In recent year, biotechnology has emerged as a frontier
branch of science increasingly being used in several
areas.
• Biotechnological approach is being employed to the
production of secondary metabolites for pharmaceutical
use.
• Plant biotechnology includes methods for tailoring plant
resources, plant cell and protoplast culture,
manipulation of nuclear and plasmid genes, plant cell
and enzyme immobilization and industrial scale
production or biotransformation.

8. Techniques of Biotransformation
 Biotransformation by immobilized cells:
• It has been observed that immobilized plant cells
may have higher production rate compared with
freely suspended cells under same conversion
conditions.
• Immobilization of plant cells as pioneered by this
technology enables entrapment of cells in a gel of
calcium alginate, polyvinyl alcohol resin on fixed
support of foam, fabric or hollow fibers.
• It creates for cells a situation which is to imitate
membership in a tissue of whole plant.
• In this, cells are expected to cease to grow and
accumulate metabolites.

9. Techniques of Biotransformation
Biotransformation by hairy root culture:
• Roots are good sources of variety of natural products like
propane alkaloid, Catharanthus alkaloids, atropine, and
hyoscyamine.
• Much attention is being given in recent years for
obtaining secondary plants products by hairy root
culture.
• The hairy roots are sub-cultured primarily on solid
medium and then on rotary shaker at 1000 rpm at 250 C
in dark in liquid medium.
• Hairy root culture is potentially applicable to the
production of all root-derived metabolites from
dicotyledonous plant.

10. Techniques of Biotransformation
Biotransformation by hairy root culture:
• Undifferentiated celli of Atropa belladonna did not
produce the tropane alkaloids-hyoscyamine but the
culture gained the ability to synthesize this compound
with the differenciation of roots.
• Whitakar produced Onion culture with high levels of
ethylcysteine sulfoxide which does not occurs naturally in
Allium’s species.

11. Techniques of Biotransformation
Biotransformation by free cells
• This is also a useful technique for biotransformation where both free
cells and immobilized cell system are useful.
• The kinds of biotransformation reaction include oxidation,
hydroxylation, methylation, and acylation.
• Following strategies need to be followed for maximal release of
secondary metabolites by cultured cells.
a) Selection of clones with high efficiency.
b) Permeabilization of cells
c) Selective removal of metabolites from media.
d) Optimization of media for maximization of production and
excretion.
• These cells are growing in different types of cultures of which two
are given here:
1. Callus culture 2. Suspension culture.

12. Callus culture
• Callus culture is a mass of cells or tissue resulted subsequent to
initiation & continued proliferation of the undifferentiated
parenchyma cells from parent tissue on a clearly defined semi solid
media.
• When an explants from a differentiated tissue is cultured on a
medium. The quiescent cells undergo changes to achieve
meristematic state.
• This phenomenon of mature cells reversion back to the
meristematic state leading to the formation of callus growth is called
differentiation.
• Moreover, the cells from callus are capable of generating into whole
plant, a phenomenon referred to as redifferentiation.
• The callus formation is controlled by the endogenous auxin &
cytokinin.
• Organogenisis can be initiated & regulated in the callus culture by
the manipulation of the ratio of auxin & cytokinins.

13. Suspension culture
• This culture essentially contains homogenous individual plant cells in its
liquid medium.
• The suspension cultures are generally initiated by transferring an established
callus tissue to an agitated liquid nutrient medium in Erlenmeyer culture
vessels.
• The composition of the medium for the establishment of suspension cultures
is same as defined for callus cultures except for the addition of agar.
• The soft callus generally forms in a suspension culture without much
difficulty. The suspension culture is usually incubated at 250C in darkness or
in low intensity fluorescent light. A cell suspension is generally formed within
4 to 6 weeks. The cells grown in culture meristematic & usually
undifferentiated & there is no evidence that cells of shoot or root origin is
metabolically different. The suspension culture is sub cultured by the transfer
at regular intervals of untreated or fractionated aliquots of the suspension to
fresh medium.
• Several forms of suspension cultures are commonly utilized as follows
• 1. Batch suspension cultures
• 2. Semi continuous culture
• 3. Continuous culture

14. Trends in Production of Secondary
Plant Metabolites from Higher Plants
• Plant cell and tissue cultures can be established routinely under
sterile conditions from explants, such as plant leaves, stems, roots,
and meristems for multiplication and extraction of secondary
metabolites. Strain improvement, methods for the selection of high-
producing cell lines, and medium optimizations can lead to an
enhancement in secondary metabolite production. The capacity for
plant cell, tissue, and organ cultures to produce and accumulate
many of the same valuable chemical compounds as the parent plant
in nature has been recognized almost since the inception of in
vitro technology.
• The strong and growing demand in today's marketplace for natural,
renewable products has refocused attention on in vitro plant
materials as potential factories for secondary phytochemical
products and has paved the way for new research exploring
secondary product expression in vitro
• There is a series of distinct advantages to producing a valuable
secondary product in plant cell culture, rather than in vivo in the
whole crop plant.

15. These include the following:
• Production can be more reliable, simpler, and more
predictable.
• Isolation of the phytochemical can be rapid and efficient,
when compared with extraction from complex whole
plants.
• Compounds produced in vitro can directly parallel
compounds in the whole plant.
• Interfering compounds that occur in the field-grown
plant can be avoided in cell cultures.
• Tissue and cell cultures can yield a source of defined
standard phytochemicals in large volumes.
• Tissue and cell cultures are a potential model to test
elicitation.
• Cell cultures can be radiolabeled, such that the
accumulated secondary products, when provided as feed
to laboratory animals, can be traced metabolically.

16. • While research to date has succeeded in producing a wide range of valuable
secondary phytochemicals in unorganized callus or suspension cultures, in
other cases production requires more differentiated micro plant or organ
cultures.[12] This situation often occurs when the metabolite of interest is
only produced in specialized plant tissues or glands in the parent plant. A
prime example is ginseng (Panax ginseng). Because saponin and other
valuable metabolites are specifically produced in ginseng roots, root culture
is required in vitro. Similarly, herbal plants such as Hypericum
perforatum (St. John's wort), which accumulates the hypericins and
hyperforins in foliar glands, have not demonstrated the ability to
accumulate phytochemicals in undifferentiated cells.[13] As another
example, biosynthesis of lysine to anabasine occurs in tobacco (Nicotiana
tabacum) roots, followed by the conversion of anabasine to nicotine in
leaves. Callus and shoot cultures of tobacco can produce only trace amounts
of nicotine because they lack the organ-specific compound anabasine. In
other cases, at least some degree of differentiation in a cell culture must
occur before a product can be synthesized (e.g., vincristine or vinblastine
from Catharanthus roseus). Reliance of a plant on a specialized structure
for production of a secondary metabolite, in some cases, is a mechanism for
keeping a potentially toxic compound sequestered. Intensive activities have
been centered on production of natural drugs or chemoprotective
compounds from plant cell culture by one or more of the following
strategies:

17. Organ Cultures for Secondary
Metabolite Production
• Fritillaria unibracteata can be rapidly propagated,
directly from small cuttings of the bulb by the technique
of organ culture. The cultured bulb can be harvested
after a 50-day culture period in MS media supplemented
with 4.44 - M BA and 5.71 - M IAA. The growth rate was
about 30–50 times higher than that under natural wild
growth conditions. The content of alkaloid and beneficial
microelements in the cultured bulbs was higher than
found in the wild bulb.[14]
• In vitro shoot multiplication of Frangula alnus was
obtained on woody plant medium with indole-3-acetic
acid and 6-benzylaminapurine, the highest metabolite
production (1731 mg/100 g of total anthraquinone was in
the shoots grown on the MS medium with addition of 1-
naphthilaceneacetic (NAA) (0.1 mg/l) and thidiazuron
(TDZ) (0.1 mg/l).[15]

18. Precursor Addition for Improvement
of Secondary Metabolite Production
• The treatment of plant cells with biotic and/or abiotic elicitors has
been a useful strategy to enhance secondary metabolite production
in cell cultures.[11] The most frequently used elicitors in previous
studies were fungal carbohydrates, yeast extract, M,J and chitosan.
MJ, a proven signal compound, is the most effective elicitor of taxol
production in Taxus chinensis Roxb.[16] and gonsenoside
production in P. ginseng C.A. Meyercell/organ culture.[17,18,19]
• The involvement of amino acids in the biosynthesis of hyperforin
and adhyperforin was reported in H. perforatum shoot cultures.
Valine and isoleucine, upon administration to the shoot cultures,
were incorporated into acyl side chain of hyperforin and
adhyperforin, respectively. Feeding the shoot cultures with
unlabelled lisoleucine at a concentration of 2 mM induced a 3-7-fold
increase in the production of a hyperforin.[20] Production of
triterpenes in leaf-derived callus and cell suspension cultures
of Centella asiatica was enhanced by the feeding of amino acids. In
the callus culture, manifold increase of asiaticoside accumulation
was reported with the addition of leucine.[21]

19. Elicitation of In vitro products
• Plants and/or plant cells in vitro show physiological and
morphological responses to microbial, physical, or chemical factors
which are known as “elicitors.” Elicitation is a process of inducing or
enhancing synthesis of secondary metabolites by the plants to
ensure their survival, persistence, and competitiveness.[11,22] The
study was applied in several abiotic elicitors to enhance growth and
ginseng saponin biosynthesis in the hairy roots of P. ginseng.[23]
Generally, elicitor treatments were found to inhibit the growth of
the hairy roots, although simultaneously enhancing ginseng saponin
biosynthesis. Tannic acid profoundly inhibited the hairy root growth
during growth period.
• The production of secondary metabolites in callus, cell suspension,
and hairy roots of Ammi majus L. is by exposing them to elicitors:
benzo (1,2,3)-thiadiazole-7-carbothionic acid S-methyl ester and
autoclaved lysate of cell suspension of bacteria- Enterobacter
sakazaki.[24] GC and GC-MS analysis of chloroform and methanol
extracts indicated a higher accumulation of umbelliferone in the
elicited tissues than in the control ones. Chitosan was the biotic
elicitor polysaccharide and it is eliciting the manifold increase of
anthraquinone production in Rubia akane cell culture.

20. Hairy Root Cultures as a Source of
Secondary Metabolites
• The hairy root system based on inoculation with Agrobacterium
rhizogenes has become popular in the two last decades as a method of
producing secondary metabolites synthesized in plant roots.[11,26] The
hairy root phenotype is characterized by fast hormone-independent growth,
lack of geotropism, lateral branching, and genetic stability. The secondary
metabolites produced by hairy roots arising from the infection of plant
material by A. rhizogenes are the same as those usually synthesized in
intact parent roots, with similar or higher yields.[27] This feature, together
with genetic stability and generally rapid growth in simple media lacking
phytohormones, makes them especially suitable for biochemical studies not
easily undertaken with root cultures of an intact plant. During the infection
process, A. rhizogenes transfers a part of the DNA (transferred DNA, T-
DNA) located in the root-inducing plasmid Ri to plant cells, and the genes
contained in this segment are expressed in the same way as the endogenous
genes of the plant cells. Some A. rhizogenes, such as strain A4, have the T-
DNA divided into two sections: the TR-DNA and TL-DNA, each of which
can be incorporated separately into the plant genome. Two sets of pRi genes
are involved in the root induction process: the aux genes located in the TR
region of the pRi T-DNA and the rol (root loci) genes of the TL region.[28]
The hairy roots are normally induced on aseptic, wounded parts of plants by
inoculating them with A. rhizogenes

21. Immobilization Scaling up of
Secondary Metabolite Accumulation
• Advances in scale-up approaches and immobilization
techniques contribute to a considerable increase in the
number of applications of plant cell cultures for the
production of compounds with a high added value. Plant-
derived compounds with cancer chemotherapeutic or
antioxidant properties use rosmarinic acid and taxol as
representative examples.
• Cell cultures of Plumbago rosea were immobilized in calcium
alginate and cultured in Murashige and Skoog's basal medium
containing 10 mM CaCl2 for the production of plumbagin, an
important medicinal compound.[36] Studies were carried to
find out the impact of immobilization on the increased
accumulation of this secondary metabolite. Immobilization in
calcium alginate enhanced the production of plumbagin by
three-, two-, and one-folds compared with that of control, un-
crosslinked alginate and CaCl2 -treated cells, respectively.

22. Elicitation:
• Elicitors are compounds of biological origin involved in plant microbe
interaction. Elicitors are considered as mediator compounds which
induce secondary metabolites formation in cells cultures.
• Varieties of elicitors have been used for production of secondary
metabolites.
• Polysaccharides in alginate are known to act like elicitor for shikonin
production in lithospermum erythrorhizen cells & for echinatin
production by glycyrrhiha echinatin cells.
• Elicitation improves the efficiency of Sec. Product accumulation in
plant cell culture by:
a) Minimizing up on time
b) Avoiding change of media
c) Induction of enzymes involved in biosynthetic pathway.
d) Inducing excretion of metabolites into the medium.

23. Techniques of Biotransformation
 Precursor feeding:
• It is another diverse, but contradictory tool for enhancing
secondary metabolite production
• There are two distinct methods of increasing the precursor supply
within the cell, firstly by addition to the medium in which care the
uptake mechanism may not be limiting.
• Secondly, by selecting for resistance to precursor analogues in
which the intracellular level may be modified for e.g. shikonin
production increase three fold when cultured cells of
Lithospeimum species are fed with L-Phenylalanin.
Similarly Datura sp. Cell suspension cultures are supplemented
with hydroquinone, in traces, the arbutin synthesis increase
considerably.

24. Application of Biotransformation
• Biotransformations of steroids:
Digitoxin: In which digitoxigenin and digitoxin
and their derivatives can be extracted from the
leaves of Digitalis lanata.
• In the process of biotransformation of Digitoxin
many compounds are produced, out of which
purpurea glycoside A is mainly produced and
methyl digitoxin is usable substrate.
• In biotransformation purpurea glycoside A is a
main product and deacetyl lanotoside C and
lanatoside C is minor product.

25. Taxol
• Taxol (paclitaxel), a complex diterpene alkaloid found in the
bark of the Taxus tree, is one of the most promising
anticancer agents known due to its unique mode of action on
the microtubular cell system Figure 1.[38] At present,
production of taxol by various Taxus species cells in cultures
has been one of the most extensively explored areas of plant
cell cultures in recent years owing to the enormous
commercial value of taxol, the scarcity of the Taxus tree, and
the costly synthetic process.[39,40] In order to increase the
taxoid production in these cultures, the addition of different
amino acids to the culture medium was studied, and
phenylalanine was found to assist in maximum taxol
production in Taxus cuspidata cultures.[41] The influence of
biotic and abiotic elicitors was also studied to improve the
production and accumulation of taxol through tissue cultures.

26. Morphine and codeine
• Latex from the opium poppy, Papaver somniferum, is a
commercial source of the analgesics, morphine, and codeine.
Callus and suspension cultures of P. somniferum are being
investigated as an alternative means for the production of
these compounds [Figure 2]. Production of morphine and
codeine in morphologically undifferentiated cultures has been
reported.[45,46] Without exogenous hormones, maximum
codeine and morphine concentrations were 3.0 mg/g dry
weight and 2.5 mg/g dry weight, respectively, up to three
times higher than in cultures supplied with hormones.
Biotransformation of codeinone to codeine with immobilized
cells of P. somniferum has been reported by Furuya et al.
(1972).[47] The conversion yield was 70.4%, and about 88% of
the codeine converted was excreted into the medium.

27. Diosgenin
• Diosgenin is a precursor for the chemical synthesis of
steroidal drugs and is tremendously important to the
pharmaceutical industry.[50] In 1983, Tal et al.[51]
reported on the use of cell cultures of Dioscorea
deltoidea for the production of diosgenin [Figure 4].
They found that carbon and nitrogen levels greatly
influenced diosgenin accumulation in one cell line.
Ishida (1988) established Dioscorea immobilized cell
cultures, in which reticulated polyurethane foam was
shown to stimulate diosgenin production, increasing the
cellular concentration by 40% and total yield by 25%.
Tal et al.[50] have been able to obtain diosgenin levels as
high as 8% in batch-grown D. deltoidea cell suspensions.

28. Camptothecin
• Campothecin, a potent antitumor alkaloid, was
isolated from Camptotheca acuminata.[58] Sakato
and Misawa[59] induced C. acuminata callus on MS
medium containing 0.2 mg/l 2,4-D and l mg/l
kinetin and developed liquid cultures in the
presence of gibberellin, l-tryptophan, and
conditioned medium, which yielded camptothecin at
about 0.0025% on a dry weight basis. When the
cultures were grown on MS medium containing 4
mg/l NAA, accumulation of camptothecin reached
0.998 mg/l.[

29. Berberine
• Berberine is an isoquinoline alkaloid found in the roots of Coptis
japonica and cortex of Phellondendron amurense. This
antibacterial alkaloid has been identified from a number of cell
cultures, notably those of C.
japonica,[61,62,63] Thalictrum spp.,[64,65] and Berberis spp.[62]
The productivity of berberine was increased in cell cultures by
optimizing the nutrients in the growth medium and the levels of
phytohormones.[63,66,67] By selecting high-yielding cell lines,
Mitsui group produced berberine on a large scale with a productivity
of 1.4 g/l over 2 weeks. Other methods for increasing yields include
elicitation of cultures with a yeast polysaccharide elicitor, which has
been successful with a relatively low-producing Thalictrum
rugosumculture.[68] The influence of spermidine on berberine
production in Thalictrum minus cell cultures has been reported by
Hara et al.

30. Application of Biotransformation
• Morphine alkaloids: Codeine is an analgesic and
cough-suppressing drug and Papaver somniferum L. is a
traditional commercial source of codeine & Morphine
which can be converted to codeine.
• Mature capsule of P.bracteatum accumulates up to 3.5 %
of the baine, which also can be converted to codeine.
• Berberine: Berberine is an isoquinoline alkaloid, which
is distributed in roots of coptis Japonica, & cortex of
philodendra’s anurans. Addition of a polyamine, sperm
dine, was found to stimulate the production of berberine
by Thalictrum minus cell suspension cultures.

31. Application of Biotransformation
• Vincristine: It is reported biotransformation of a
hydro vincritin to vindolin by a cell extract of
Catharanthus-roseus cell suspension culture.
Their species produce high-value specialist;
phytochemicals, highly valuable drugs like
vinblastin & Vincristine that are used for the
treatment of cancer are extracted from
Catharanthus roseus plants.

32. Biotransformation of terpenoids:
• Monoterpenes : Biotransformations have been demonstrated
with mentha cell lines capable of transforming pulegon to
iromenthone, & (-)- Mentone to (+) neomenthal.
• Biotransformations of paclitaxel (Taxel) by plant cell culture:
Incubation of eucalyptus citriodera, Azadirachata indica &
capsicum annum cell cultures with pacitaxel was carried in a
refrigerated shaker incubator at 120 rpm & 25oC (±1 oC) for
48 hr. The culture were extracted analyzed by HPLC. Only E.
citriodora cultures were able to biotransform paclitaxel into
two known compound (baccatin III & deacetyl baccatin III) &
an unknown compound.

33. Factors affecting biotransformation:
• Biotransformation is longely depends an various
factors like physiological, biochemical aspects &
environmental conditions of cell culture.
• Source of origin of plant tissue, culture media
formulation, carbon sources, plant growth
regulator, physical condition of cultures like
oxygen, co2 influence the production of sec.
metabolites

34. • Origin of plant tissue: Production of secondary
product in cultures in under the control of genetic
nature of the explants. Scientist proved that culture
derived from high fielding cultivars, produced high
nicotine content, where as cultures established from
low fielding cultivate showed less potential for
nicotine production.
• Culture Media: Chemical composition of culture
media for establishing callus or suspension culture
influences the production of biomass & also the
synthesis of sec. Metabolites. An ideal culture
conditions are to be maintained to achieve plant
products in culture by manipulating an optimum
balance between biomass production & secondary
metabolite production.

35. • Growth regulators: Growth regulators affect growth &
synthesis of sec. Metabolites of cultured cells. Sometime
combination of auxins & cytokinins had synergistic effect
while other had antagonistic influence on steroidal
synthesis. Taxol producing plant, Taxus cuspidate was
significantly promoted by addition of gibberellic acid into
the solid medium.
• Carbon source in the medium: Sucrose is the most
commonly used carbon source in the culture medium for
the growth of tissue, & also drastically influences, the
biosynthesis of sec. Metabolites in culture. Many other
carbon compounds like glucose, fructose, and galactose
also influence product accumulation in cultures. Out of all
the carbon smokes used so for in cultures, sucrose &
glucose gave the most encouraging results in terms of
biomass production of product field.

36. • Temperature: Plants are frequently able to exist in a
considerable range of tem. In general, the formation
of volatile oils appeases to be enhanced at higher
temperature.
• PH: A medium PH is usually adjusted to between 5 &
6 before autoclaving & extreme of PH are avoided.
• Light intensity: Light intensity stimulate the in vitro
process of enzyme action. High light intensity
suppressed the production of nicotine in Nicotiana
tabacum, where as continuous growth in darkness
enhanced nicotine synthesis & its accumulation.
Light is a factor, which helps to detamine the amount
of glycosides, or alkaloids produced with belladonna,
Stramonium & cinchona ledgeriana full Sunshine
gives a higher content of alkaloid than does shade.

37. • High cell density culture: To increase the
productivity of sec. Metabolites, high cell
density culture have been investigated,
using a nearly designed fermented &
optimized culture medium, copies
Japonica cells were grown up to 759L of
cell mass.

38. • High cell density culture: To increase the
productivity of sec. Metabolites, high cell
density culture have been investigated,
using a nearly designed fermented &
optimized culture medium, copies
Japonica cells were grown up to 759L of
cell mass.