Plant cell and organ cultures can be used to produce valuable secondary metabolites. Three main approaches were discussed: 1) using plant cell and organ cultures, 2) using microbial cell factories, and 3) using molecular biopharming. Plant cell and organ cultures involve growing plant cells, tissues, or organs in vitro on nutrient media. This allows mass production of metabolites and can help address issues with traditional extraction. Microbial cell factories and molecular biopharming use genetically engineered microbes or organisms to produce metabolites. Taxol is an important anticancer drug produced through plant cell cultures due to supply issues with traditional extraction from yew trees.

1. Dr. Refaat Hamed
Monday, 14 September 2015
Biotechnological Production of
Natural Products (2ry Metabolites)

2. Biotechnology??
“Any technological application that uses biological systems,
living organisms or derivatives thereof, to make or modify
products or processes for specific use”
(UN Convention on Biological Diversity,Art. 2)
Why Biotechnology??
• Current world population ~6.8 billion and expected do double by
2050.
• The challenge for the future lies in global food security that
necessitates a doubling of food production by 2050!!
• According to WHO, ~80% of people worldwide rely mainly on
traditional/herbal medicines for their 1ry healthcare needs.
• Biotechnology can be used to feed, heal and biofuel the world!!

3. Biotechnological Production of Natural Products
Three biotechnological approaches for production of natural products
will be briefly covered:
1. Using Plant Cell and Organ Cultures
2. Using Microbial Cell Factories
3. Using Molecular Biopharming
• The production of 2ry metabolites via living cell factories (plant,
bacteria or fungi), in a bioreactor, is an economic and echo-friendly
alternative to traditional approaches such as:
a- chemical synthesis
b- extraction from over-collected plant species
• These renewable approaches can also benefit from genetic
transformation techniques to enable the combinatorial
biosynthesis of novel products.

4. Plant Cell and Organ Cultures
Plant cell culture is similar in concept to ‘Dolly the sheep’
but using plants.
1- Definition
2- History
3-Theory
4- Materials and methods
5- Applications

5. Plant Cell and Organ Cultures: Definition
• Plant tissue culture refers to growing and multiplication
of cells, tissues and organs of plants on defined nutrient
media under aseptic and controlled conditions. The
part(s) used (explant) can be seeds, embryos, organs,
tissues, cells or plant protoplasts.
• The growth takes place outside an intact plant (in vitro
culture).
• To culture cells/organs from a specific plant for the first
time is a matter of trial and error (i.e. you have to
experiment with different culture conditions).

6. Plant Cell and Organ Cultures: History
Landmark discoveries:
• 1902: Haberlandt proposed the concept of in vitro cell culture
• 1904: Hannig cultured embryos from several cruciferous species
• 1922: Kolte and Robbins successfully cultured root and stem tips
respectively
• 1926: Went discovered first plant growth hormone (Indole-3-
acetic acid, IAA)
• 1934: White introduced vitamin B as growth supplement in tissue
culture media for tomato root tip
• 1939: Gautheret, White and Nobecourt established successful
proliferation of callus cultures

7. Plant Cell and Organ Cultures: Theory
Living cells are “totipotent”, which means that every cell
contains all genetic information to grow a new plant. Plant
cell and tissue culture exploit this characteristic for, e.g.,
micropropagation of plants. One may initiate cell cultures
from any part of the plant by growing an explant on a
suitable medium supporting cell growth.

8. Plant Cell Culture: Methodology
• A callus is initiated from pieces of tissue cut from
surface-sterilized plants. The explants are placed on
different solid growth media; in successful media, callus
tissue will appear on the explants in the course of 2–6
weeks.
• A callus is subsequently cut from the explant and further
subcultured. Callus material can be inoculated in liquid
medium (in flasks), and cell suspension cultures can be
obtained through continuous shaking.
• The whole process (from plant to a stable cell
suspension culture) may take 6–9 months. It is essential
to let a cell culture first stabilize before studying the
production of desired compounds.

9. Scheme of the procedure of
initiation and maintenance of
plant cell suspension cultures.
Growth measurement of plant
cell suspension culture.

10. Plant Organ Culture:
Young leaf parts isolated from
garlic cloves that served as
explants showing the
initiation of callus formation
after one week on callus
induction medium (A and B).
Sufficient garlic callus growth
after 6-8 weeks on MS6
medium (A and B).
Garlic callus growing on
medium at pH 4.5 and
showing development of few
roots and small green sectors
(A and B).
• Callus tissue consists of two different group of cells; soft parenchyma
cells and meristematic cells, which eventually proliferate and undergo
morphogenesis, in the presence of the right phytohormone.

11. Plant Cell and Organ Cultures: Requirements
I- Plant material II- Growth Media III- Equipment

12. Plant Cell and Organ Cultures: Requirements
I- Plant material II- Growth Media III- Equipment
• The part of the plant used (known as explant) can be small piece of
a stem, leaf, flower, seed, root, cotyledon or any other living
tissue/cell(s).
• Micropropagation refers to the application of plant tissue culture
techniques to clone (propagate) a certain species from its explant.
• Younger and fresh explants are preferred.
• Explants must be initially sterilized (using 70% ethanol and/or fresh
diluted sodium hypochlorite) to remove micro-organisms.
• Tissue selection of the explant is very important and depends on a
number of factors, including the purpose of the culture.
• Dicotyledones are more ameable to callus induction than
monocotolydones.

13. Plant Cell and Organ Cultures: Requirements
I- Plant material II- Growth Media III- Equipment
The nutrient medium used for plant tissue culture should consist of:
1. Inorganic salts (macro and micro-elements)
2. Carbon source (2-4% sucrose or glucose)
3. Organic supplements (e.g. peptone and yeast extracts)
4. Vitamins (thiamine-HCl, nicotinic acid, myo-inositol and others)
5. Growth regulators/Phytohormones/Elicitors
• Abscisic acid
• Gibberellins
• Auxins: e.g. Indole-3-acetic acid (IAA), often induce adventitious roots.
• Cytokinins: e.g. kinetin and zeatin, often induce adventitious buds/shoots.
• Jasmonate, ethylene and salicyclic acid

14. Plant Cell and Organ Cultures: Requirements
I- Plant material II- Growth Media III- Equipment
• Agar or gelatin can be added (as solidifying agents) if a solid medium
is required (mainly for establishment and maintenance of callus
cultures). Liquid nutrient media are used to support the growth of
plant cell suspension cultures.
• Murashige and Skoog medium (MS Medium) is one of the most
commonly used media for plant tissue culture. It contains high
concentration of nitrate, potassium and ammonia.
• Another commonly used medium is B5 medium, which contains
lower levels of inorganic nutrients compared to MS medium.
• The media/additives employed can be sterilized either via autoclaving
or filtration (if thermo-labile).
• Selection of the optimum nutrient composition is a matter of trial and
error (unless already reported).

15. Plant Cell and Organ Cultures: Requirements
I- Plant material II- Growth Media III- Equipment
The following are required:
1. Laminar air flow cabinet

16. Plant Cell and Organ Cultures: Requirements
I- Plant material II- Growth Media III- Equipment
The following are required:
1. Laminar air flow cabinet
2. Autoclave

17. Plant Cell and Organ Cultures: Requirements
I- Plant material II- Growth Media III- Equipment
The following are required:
1. Laminar air flow cabinet
2. Autoclave
3. Oven
4. Equipment for sterilization by filtration

18. Plant Cell and Organ Cultures: Requirements
I- Plant material II- Growth Media III- Equipment
The following are required:
1. Laminar air flow cabinet
2. Autoclave
3. Oven
4. Equipment for sterilization by filtration
5. Water distillation apparatus
6. Culture room: incubator with light and temperature control

19. Plant Cell and Organ Cultures: Requirements
I- Plant material II- Growth Media III- Equipment
The following are required:
1. Laminar air flow cabinet
2. Autoclave
3. Oven
4. Equipment for sterilization by filtration
5. Water distillation apparatus
6. Culture room: incubator with light and temperature control
7. Shelves, shakers and centrifuges
8. Equipment for media preparation (balance, pH meter, ….)
9. Fermenter or Bioreactor: for scaling-up cell suspension culture

20. Plant Cell and Organ Cultural Parameters
The following culture parameters can affect culture growth and
subsequently the levels of secondary metabolites production:
1. Temperature: 25±2 °C is suitable for most cultures
2. Light: e.g. higher content of shikonin-type naphthaquinones in
Lithospermum erythrorhizon cultures when grown in the dark.
3. Plant age and origin of the callus
4. Culture media and culture technique: solid or liquid (stationary
or agitated); batch, continuous or semi-continuous,….
5. Additives (e.g. vitamins, amino acids, growth regulators, …)
6. Addition of precursors (e.g. coniferin in case of the cell suspension
culture of Podophyllum hexandrum -----> higher podophyllotoxin levels)

21. Applications: 1- Hairy/Transformed Root Culture
Many products of interest are synthesized in organized tissues, but not
formed in suspension or callus culture. Therefore, most attention has
been focused on root cultures.
Concept: The bacterium Agrobacterium rhizogenes that contains root
inducing plasmids (called Ri-plasmids) can infect plant roots and cause
them grow very fast.The resulting roots are:
• Easy to culture in artificial media without hormonal requirement
• Have high growth rate (compared to untransformed roots)
• Genetically and biochemically stable.
Applications:
1. Plant metabolic studies:
2. Phytoremediation:
Restoring environmental balance through plantation to remove
contaminants (e.g.Arsenic using sunflower)

22. Applications: 1- Hairy/Transformed Root Culture
3. Genetically transformed cultures:
The Ri-plasmid of Agrobacterium rhizogenes can be genetically engineered
to to introduce new gene/cluster of genes for transformation of the plant
to produce novel 2ry metabolites/recombinant proteins (Molecular
Biopharming).
4. Plant propagation:
To conserve/propagate endangered plant species

23. Summary
Plant
Callus
Somatic embryo cells
Regenerated roots and shoots
Regenerated plants
De-differentiation
(undifferentiated cells)
Re-differentiation
Regeneration
Cell
suspension
culture
2ry
Metabolites

24. Biotechnological Production of Natural Products: Lecture II
Alternative biotechnological approaches for production of medicinally
active 2ry metabolites include:
1. Using Plant Cell and Organ Cultures.
2. Using Microbial Cell Factories.
Ex. Microbial production of Artemisinin (antimalarial drug).
3. Using Molecular Biopharming/Biofarming
• Molecular farming is a type of production system which uses genetically modified
organisms (GMO) to produce valuable pharmaceuticals for humans and animals.
• Ex. (1): Engineering the provitamin A (β-carotene) biosynthetic pathway into
(carotenoid-free) rice endosperm.
• Ex. (II): Employment of silkworms as a “mini-factory” for the production of
pharmaceutical proteins.

25. Biotechnological Production of Natural Products: Lecture II
Alternative biotechnological approaches for production of medicinally
active 2ry metabolites include:
1. Using Plant Cell and Organ Cultures.
2. Using Microbial Cell Factories.
Ex. Microbial production of Artemisinin (antimalarial drug).
3. Using Molecular Biopharming/Biofarming
• Molecular farming is a type of production system which uses genetically modified
organisms (GMO) to produce valuable pharmaceuticals for humans and animals.
• Ex. (1): Engineering the provitamin A (β-carotene) biosynthetic pathway into
(carotenoid-free) rice endosperm.
• Ex. (II): Employment of silkworms as a “mini-factory” for the production of
pharmaceutical proteins.

26. Applications of Plant Tissue Culture Technique
1. The development of a commercial production facility for
expensive medicaments e.g. taxol.
2. The discovery of new metabolites and analogues of known
ones.
3. The selection of superior strains of medicinal plants.
4. Elucidation of the biosynthetic pathways of certain
metabolites.
5. The improvement of medicinal plants species via genetic
engineering.

27. Applications of Plant Tissue Culture for Production of
Secondary Metabolites of Therapeutic Value
1. Anticancer drugs e.g. taxol, vinblastine, vincristine,
podophyllotoxin and camptothecin
2. Cardiovascular drugs e.g. cardenolides/cardiac glycosides
(Digitalis lanata and D. purpurea). Addition of progesterone to callus
culture of D. purpurea, resulted in accumulation of digoxin and digitoxin.
3. Tonics e.g. ginsenosides (Panax ginseng). Exposure of callus culture to γ-
rays resulted in accumulation of good levels of saponins.
4. Antispasmodics e.g. furanchromones (Ammi visnaga). Exposure of
callus, cell suspension and hairy root cultures to acetylsalicyclic acid, jasmonic acid
and silicon dioxide resulted in good levels of furanochromones and
pyranocoumarins.
5. Food additives e.g. shikonin (Lithospermum erythrorhizon). Addition
of adsorbents (such as XAD-2, XAD-4 and charcoal) to the root culture resulted
in higher levels shikonin production.

28. • The National Cancer Institute (NCI), screened over 35000 plants for
anticancer activity between 1960 and 1986, and over 2000 crystalline
plant-derived compounds were isolated and tested for activity.
Anticancer drugs:
• Approximately 60% of all
drugs in clinical trials for
the treatment of cancer
are either natural products,
derived from natural
products, or contain
pharmacophores derived
from natural products.

29. Anticancer drugs: Taxol (Paclitaxel)

30. Anticancer drugs: Taxol (Paclitaxel)
• Taxol, a complex diterpene amide, was
first isolated from the bark of Taxus
brevifolia (pacific yew tree) after initial
studies at 1962, and its structure was
elucidated at 1971. Taxus brevifolia
contains low amounts of toxic alkaloids
(taxine A and B) and reasonable levels
of taxol compared to other Taxus
species.
• Taxol and taxol analogues are the most
important drugs for treatment of drug-
refractory ovarian cancer as well as
lung and breast cancers.

31. Taxol promotes the polymerization of tubulin heterodimers to microtubules.
At clinically relevant concentrations, taxol binds to microtubules resulting in
their stabilization via suppressing their dynamic changes. Taxol thus interferes
with the formation of mitotic spindle, which causes the chromosomes not to
segregate, and consequently mitotic arrest.
Anticancer drugs: Mechanism of Action of Taxol
Microtubule
(polymerized tubulin)

32. Anticancer drugs: Mechanism of Action of Taxol
Taxol promotes the polymerization of tubulin heterodimers to microtubules.
At clinically relevant concentrations, taxol binds to microtubules resulting in
their stabilization via suppressing their dynamic changes. Taxol thus interferes
with the formation of mitotic spindle, which causes the chromosomes not to
segregate, and consequently mitotic arrest.

33. Anticancer drugs: Taxol supply crisis
• Taxus species (pacific yew trees) are scarce and thus
environmentally protected due to their slow growth (6-10
years to grow).
• The content of taxol is generally very low in Taxus species.
The bark of T. brevifolia is very thin.
• Semisynthetic approaches for production of taxol from
10-deacetylbaccatin III (more abundant precursor in T.
baccata,) were initially developed for commercial
production (however the supply of 10-deacetylbaccatin III
is also limiting, and the overall process is expensive).
• Currently, plant cell culture is the major source for
commercially available taxol.

34. Taxol biosynthesis: The pathway
comprises four steps:
• Supply of geranylgeranyl diphosphate
(GGPP, the universal intermediate of
diterpenoids) via a non-mevalonate
pathway, as catalysed by GGPP
synthase.
• Taxane-ring formation as catalysed by
taxadiene synthase.
• Formation of baccatin III (an
important intermediate in taxol
biosynthesis, many P450
oxygenases are involved).
• Introduction and modification of the
phenylisoserine side chain at C-13
of the taxane ring.

35. Taxol Production via Plant Tissue Culture
• Bristol-Myers Squibb (BMS), in collaboration with Phyton, Inc.,
announced in 2003 that it is producing taxol by plant cell culture
methods (details not released, but is known to be based on cultures
of T. chinensis). The overall method is reported to be echo-friendly and
involves the use of five organic solvents.
• Production of taxol is enhanced by elicitation with methyl jasmonate
(MeJA) and other elicitors, and yields of ~295 mg/L have been
reported. There are two main regulatory steps in taxol
biosynthesis in Taxus cell suspension cultures; the taxane ring-
formation step and the C-13 side chain-introduction step. MeJA
addition upregulates 9 genes in taxol biosynthesis.
• Factors that can be manipulated to increase taxol levels include gas
composition (O2, CO2 and ethylene), osmotic pressure, and
conditioned medium; bioprocessing strategies, feeding of precursors
or sugars.

36. Taxol Production via Plant Tissue Culture
• Sucrose feeding during stationary phase results in higher cell growth and
higher taxol levels from Day 27 Day 42.
• The accumulation of taxol in cells leads to feedback repression and
product degradation. Therefore, the in situ solvent extraction (two-phase
culture) of paclitaxel from suspension cultures is essential for improving
productivity. Addition of 10% (v/v) dibutyl-phthalate during the late-log
phase improved taxol production and release with minimal inhibitory
effect on cell growth.
• Bioreactors of up to 75,000 L are being employed by ESCAgenetic (USA),
Phyton (USA), Samyang Genex (Korea), and Phyton Biotech (Germany).
• Overall, combination of the above findings resulted in >100 fold
improvement in taxol levels compared to that reported in the first patent
concerning taxol production employing suspension cultures. Genetic and
metabolic engineering efforts have the potential to further improve the
levels of taxol and other valuable products.

37. Anticancer drugs: Podophyllotoxin
• Podophyllotoxin is an antiviral and antitumor lignan obtained from the
roots and rhizomes of Podophyllum species.
• It is effective against skin cancer. Its semisynthetic derivative etoposide
is effective against brain tumor, lymphosarcoma and Hodgkin’s disease.
• Lignans are biosynthesized through the shikimic acid pathway and
formed by the union of two phenylpropane units.

38. Podophyllotoxin Mechanism of Action
• The antineoplastic activities of podophyllotoxin analogues include
preventing the assembly/polymerization of tubulin into microtubules
and inhibiting the catalytic activity of DNA topoisomerase II.

39. Podophyllotoxin Biosynthesis and Tissue Culture
• At present, the commercial source for podophyllotoxin relies on the
endangered Podophyllum genus. Therefore, the search for an alternative
renewable and economic route is imperative.
• Podophyllotoxin biosynthesis originates from phenylalanine, which gets
converted to coniferyl alcohol by various enzymes. Two molecules of
coniferyl alcohol are proposed to condense to eventually give
deoxypodophyllotoxin (the precursor for podophyllotoxin and
analogues).
• The callus culture of P. peltatum can produce podophyllotoxin. Root
culture of Linum species can produce podophyllotoxin and its 6-methoxy
derivative. Hairy root cultures of Linum species can produce 6-
methoxypodophyllotoxin and its glucoside derivatives.
• Cross-species coculture systems using P.
hexandrum cell suspension culture and Hairy
root cultures of Linum species can significantly
increase podophyllotoxin production.

40. Podophyllotoxin Biosynthesis and Tissue Culture
• Addition of a complex precursor (coniferyl alcohol and β-cyclodextrin)
to the cell suspension culture of P. hexandrum resulted in 0.013%
podophyllotoxin (dry wt. Cells) but without precursor cells produced
0.003%).
• β-D-glucoside of coniferyl alcohol (coniferin) was a better precursor but
not commercially available.

41. Anticancer drugs: Vinca Alkaloids
• Vinblastine and vincristine are indole-indoline dimeric alkaloids.
R= CH3, Vinblastine
R= CHO, Vincristine
N
N
H
OH
CO2CH3
N
N
CH3O
H
H OH
OCCH3
OCO2CH3
R
Vindoline
N
N
CH3O
H3C
H
H OH
OCCH3
OCO2CH3
Catharanthine
N
N
CO2CH3
H

42. Anticancer drugs: Vinca Alkaloids
• Vinblastine and vincristine are highly valued drugs in cancer
chemotherapy due to their potency against various types of leukemia,
Hodgkin’s disease and solid tumors.
• They are produced commercially by extraction from Catharanthus
roseus but their levels are very low (0.0005% dry wt).
Vinblastine

43. Vinca Alkaloids Mechanism of Action
• The antiproliferative activity of vinca was shown to be due to their
inhibition of assembly /polymerization of tubulin into microtubules,
and consequently, inhibiting cells from undergoing division. They bind
to tubulin at different sites from those of colchicine and taxol.

44. Production of Vinca Alkaloids
• Collaboration between National Research Council of Canada and
University of British Columbia, as well as later efforts by Mitsui
Petrochemical (Japan) established the production of vinblastine as follows:
1. Production of catharanthine by plant cell culture/fermentation
After 2-3 weeks of growth on MS medium + 3% sucrose + 1 mg/L
naphthaleneacetic acid (NAA) + 0.1 mg/L kinetin. Inducers such as vanadyl
sulfate, sodium chloride and abscisic acid can also increase the levels of
catharanthine (230 mg/L/week).
2. Formation of vinblastine via Chemical or enzymatic ligation of
catharanthine and vindoline
• Vindoline can be produced by extraction from intact C. roseus (0.2% dry
wt.).
• The enzyme for ligation is obtained by addition of 70% ammonium sulfate
to C. roseus suspension culture, which results in precipitation of the crude
enzyme.

45. Production of Vinca Alkaloids
• The crude enzyme when incubated with catharanthine and vindoline (pH=
7, 30 °C, for 3 hours) catalyses the formation of anhydrovinblastine as the
major product. Addition of excess amount of sod. borohydride after
incubation results in the formation of vinblastine.
• The coupling/ligation reaction can be achieved without the enzyme by
addition of ferric chloride and is improved by addition of oxalate and
maleate.

46. Anticancer drugs: Camptothecin
• Camptothecin (CPT) is a cytotoxic quinoline alkaloid produced by
Camptotheca acuminata and has potent antitumor activity against GIT-
cancer.
• CPT inhibits the DNA enzyme topoisomerase I (topo I). Binding of
CPT to topo I and DNA complex results in the formation of a stable
ternary complex, which This prevents DNA re-ligation and therefore
causes DNA damage and eventually results in apoptosis.

47. Camptothecin Tissue Culture
• Callus cultures from the stems of C. acuminata were initiated on solid
medium containing 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D)
and 1 mg/L kinetin.
• Induced callus was subcultured on liquid MS medium containing
gibberellin and L-tryptophan for 15 days, which resulted in
camptothecin production in low conc. (1/20 of that of intact plant).
• Addition of 4 mg/L of naphthaleneacetic acid (NAA) resulted in
higher conc. of camptothecin.

48. Microbial Production of Artemisinin
• Using synthetic biology, the metabolism of the microbe is engineered to produce
artemisinic acid, which is then secreted and can be purified from the culture media.
• Artemisinic acid is chemically converted to artemisinin and analogues thereof for
use in artemisinin-based combination therapies (ACTs) for malaria treatment.

49. Engineering the provitamin A (β-carotene) biosynthetic
pathway into (carotenoid-free) rice endosperm: Golden Rice
• Recombinant DNA technology was used to improve rice endosperm
nutritional value via a combination of transgenes enabled biosynthesis of
provitamin A.
• Provitamin A deficiency kills 670,000 children each year.
• Overall, biotechnology has the potential to make our lives easier!!