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According to WHO survey nearly 70% -
80% of total world population depends upon
herbal drugs.
The production of specialty chemicals by
plants is a multibillion industry.
WHAT ARE SECONDARY
METABOLITES?
They are the phytochemicals that do not
participate in plant metabolism.
They are not directly needed by plants as they
don’t perform any physiological functions.
They may include pharmaceuticals, flavors,
fragrances, cosmetics, food additives, feed stocks,
antimicrobials.
WHY IN VITRO?
Compounds can be produced under controlled
conditions as per market demands.
Independent of environmental factors.
Quality will be consistent as the products are
formed by a specific cell line.
Easy recovery strategies can be applied
Novel products can be produced via mutant cell
lines.
Biotransformation can be done.
MAJOR ADVANTAGES
Compounds can be produced under controlled conditions as per
market demand.
Culture system is independent of envt. Factors.
Cell growth can be controlled to facilitate improved product
formation.
Quality of product will be consistent as it produced by a specific
cell lines.
Recovery of product will be easy.
Mutant cell lines can be developed for the production of novel
compounds of commercial importance which are not normally
found in plant.
Production time is less and labor cost are minimal.
Biotransformation
MAJOR LIMITATIONS:
Lower quantity in comparison to field plants.
Lower quantity in comparison to permanent tissue.
Cultured cells are genetically unstable, so,
susceptible to mutation.
Aging of the culture adversely affect the
production level.
Vigorous stirring may damage the cells.
WHY DO PLANTS PRODUCE 2°-
METABOLITES?
For protecting themselves from infections by
producing some antimicrobial compounds called
phytoalexins.
It is believed that production of 2°-metabolites is
linked to the induction of morphological
differentiation.
It is often seen that some of differentiated tissues get
specialized to produce specific 2°-metabolites, and in
comparison to in vitro cultures, which are the masses of
undifferentiated cells, produces higher level of 2°-
metabolites.
APPLICATIONS OF 2°-
METABOLITES:
Chemically they may be alkaloids, terpenoids,
glycosides etc.
Pharmaceuticals, flavors, perfumes,
agrochemicals, insecticides and raw materials
for industries.
Shikonine-A dye from Lithospermum
erythrorhizon.
Production of 2°-metabolites is a multibillion
industry.
One kg of vincristine and vinblastine cost
Name of the 2°-Metabolites Pharmaceutical Uses
Codenine Analgesics
Quinine Anti-malarial
Atropine Muscle relaxants
Digoxin Treatment of cardiovascular
disorders
Reserpine Hypotensives
Jasmine Perfumes
Pyrithrins Insecticides
Stevioside Food sweeteners
Vincristine Anticancer agents
STRATEGIES FOR PRODUCTION
OF 2°-METABOLITES IN VITRO
Selection of cell lines for high yield of
secondary metabolites.
Large scale cultivation of plant cells
Medium composition and effect of nutrients
Elicitor induced production of sec. met.
Effect of envt. Factors.
Biotransformation using plant cell culture.
Secondary metabolites release and analysis.
I. SELECTION OF CELL LINES FOR HIGH
YIELD:
Separation of producer cells from the non-
producer ones.
Euphorbia mili – anthocyanin- Yamada and
Fujita (1973)
Shikonin-
Not all cell types produce the
desired metabolite
Within a specific cultivar of Catharanthus
roseus, 62% of the clones produced the
desired metabolite
whereas in another only 0.3% produced the
metabolite
Culture conditions must be
optimized
e.g. concentrations of sugar, hormones, and
vitamins
light
temperature
Metabolite production is frequently
higher in cell cultures
Berberine production from Coptis japonica
is about 5% of dry weight after 5 years of
root growth, which equals 0.17 mg/g per
week.
Whereas in selected cell lines it can be
13.2% of the dry weight in cell culture after
3 weeks, which is about 44 mg/g/week or
about 250 times higher
Many secondary metabolites are
produced in roots
Scientists have developed a form of root culture
using Agrobacterium rhizogenes, the cause of
hairy root disease. (Show Fig 14.3)
Cells transformed with some of the bacteria’s
DNA, causes the cells to be more sensitive to
the hormones they produce. The cells form into
roots. These roots grow very fast and produce
the secondary metabolites that ordinary roots
produce.
Root cultures are often better
than cell cultures
Roots often secrete the metabolites into the
surrounding medium, making it easy for
collection.
Charcoal can be added to the medium, the
metabolites are absorbed by the charcoal,
and this stimulates even higher production
of the metabolite.
For optimal production of secondary
metabolites a two medium approach is
desirable.
First medium: reqd for good growth of cells.
Second medium: refer to as production
medium promotes secondary metabolites
production.
Effect of precursors
Addition of precursors to the medium
enhances product formation.
Eg: ornithine, phenylalanine, tyrosine and
sodium phenylpyruvate, precursors
typtamine and secologanin increase
ajmalicine production in C.roseus culture.
Production- very low, demand- not met…
Effort : for product formation at molecular
level, and exploit the ways for increased
production.
Elicitors are the compounds of biological
origin which stimulate the production of
secondary metabolites, and the phenomenon
is called ELICITATION.
ELICITORS
ENDOGENOUS EXOGENOUS
ABIOTICBIOTIC
All elicitors
of biological
origin
Physical agent:
heat, cold, UV,
osmotic pressure
Chemical agent:
antibiotics,
fungicide, etc..
Produced by
microbes. Eg:
chitin, chitosan,
glucans.
Within plant cell:
pectin, pectic acid,
cellulose, etc
METHODOLOGY FOR ELICITATION
a) Selection of microorganisms
b) Co-culture
Effect of light
Effect of incubation temp
Effect of pH
Aeration of culture
Conversion of one chemical into another by
using biological system as biocatalyst is
regarded as biotransformation or
bioconversion.
Conversion of some less imp substances to
valuable medicinal or commercially
important products.
Bioconversion may involve many reaction
eg: hydroxylation, reduction, glycosylation.
Good eg is : large scale production of cardiovascular
drug digoxin from digitoxin by Digitalis lanata.
Cell culture Digitalis purpurea or Stevia rebaudiana
can convert steviol into steviobiocide and steviocide
which are 100 times sweeter than cane sugar,
For secondary metabolites stored in vacuoles of
cells, two membranes have to be disrupted.
Permeabilizing agents such as DMSO can be
used for the release of products.
Separation and purification is costly, so two
approach are made:
a) Production of sec. met. Should be high as
possible
b) Formation of side product which interfere must
be minimal.
Kinds of Secondary Metabolites
alkaloids
phenolics (including polyphenols and
tannins)
terpenoids
Precursors can be fed to either cell culture or
roots to produce the metabolite in question.
In addition, cells can be genetically
engineered to over-produce the metabolite,
but this may be more difficult with pathways
that have many enzymes.
Some secondary metabolites
produced in cell and root
culture
L-DOPA: a precursor of catecholamines, an
important neurotransmitter used in the
treatment of Parkinson’s disease
Shikonin: used as an anti-bacterial and anti-
ulcer agent
Anthraquinone: used for dyes and medicinal
purposes
Opiate alkaloids: particularly codeine and
morphine for medical purposes
Berberine: an alkaloid with medicinal uses
for cholera and bacterial dysenterry
Valepotriates: used as a sedative
Ginsenosides: for medicinal purposes
Rosmarinic acid: for antiviral, suppression
of endotoxin shock and other medicinal
purposes
Quinine: for malaria
Cardenolides or Cardioactive glycosides: for
treatment of heart disease
Taxol: an example
Taxol is a unique anticancer drug from the
bark of the Pacific Yew (Taxus breviola)
Pacific Yew Facts
Pacific Yew was considered a trash tree by
foresters
The tree is slow growing, taking about 50
years to mature
It grows best in the understory of other trees,
not doing well in direct sunlight
Taxol Facts
Very effective treatment against ovarian
cancer, breast cancer, melanoma, and colon
cancer
Stops cell division, thus blocking cancer. It
does this by interfering with microtubule
function. Microtubules are responsible for
pulling apart the sets of chromosomes in
mitosis.
Taxol Needs
It is estimated that 250 kg of pure Taxol are
needed to treat cancer in the USA. This would
require the bark of about 360,000 trees per
year!
Obviously Taxol woud be very expensive by
this method (approximately $200,000 to
$300,000 per kg).
Taxol is a very good target for
biotechnology
a) tissue culture of bark cells
b) fungus produces taxol
c) alternative species
d) genetic engineering
e) chemical synthesis
a) tissue culture of bark cells
Many cells from different bark tissues from different
trees were screened.
There are at least 25 fold differences in production.
It was found to be secreted into the medium thus
facilitating collection.
So far 1 to 3 mg of taxol are produced per liter of
cell culture. This is equivalent to about 25 g of bark.
b) fungus produces taxol
It was found that a fungus that colonizes
yew trees also produces taxol
Fungal culture technology which is better
developed than plant cell culture technology
could be an important source for taxol
production
c) alternative species
Some researchers found that the European
Yew (Taxus baccata) produces a precursor
to taxol.
This precursor can then be converted to an
analog of taxol in the laboratory.
The precursor is used for chemical synthesis
of taxol.
d) genetic engineering
Other scientists are trying to identify and
clone the genes which produce taxol
This will enable them to scale up production
in cell culture
e) Chemical synthesis
Until 1994, chemical synthesis was
formidable
3 different ways to synthesize taxol are now
known
Some take up to 13 steps
Cost per patient still expensive; about
$20,000
This is because it usually takes 10 years of research to
produce a product. This requires that a product sell for at
least $400 per kg to make it economically worthwhile.
Many of these Third World countries may
lose market share to superior, more efficient
production of secondary metabolites in
industrial countries.
Is this right? Is it fair? Are third world
countries capable of competing? What
should they do?
THANKU

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Production of secondary metabolites

  • 1.
  • 2. According to WHO survey nearly 70% - 80% of total world population depends upon herbal drugs. The production of specialty chemicals by plants is a multibillion industry.
  • 3. WHAT ARE SECONDARY METABOLITES? They are the phytochemicals that do not participate in plant metabolism. They are not directly needed by plants as they don’t perform any physiological functions. They may include pharmaceuticals, flavors, fragrances, cosmetics, food additives, feed stocks, antimicrobials.
  • 4. WHY IN VITRO? Compounds can be produced under controlled conditions as per market demands. Independent of environmental factors. Quality will be consistent as the products are formed by a specific cell line. Easy recovery strategies can be applied Novel products can be produced via mutant cell lines. Biotransformation can be done.
  • 5. MAJOR ADVANTAGES Compounds can be produced under controlled conditions as per market demand. Culture system is independent of envt. Factors. Cell growth can be controlled to facilitate improved product formation. Quality of product will be consistent as it produced by a specific cell lines. Recovery of product will be easy. Mutant cell lines can be developed for the production of novel compounds of commercial importance which are not normally found in plant. Production time is less and labor cost are minimal. Biotransformation
  • 6. MAJOR LIMITATIONS: Lower quantity in comparison to field plants. Lower quantity in comparison to permanent tissue. Cultured cells are genetically unstable, so, susceptible to mutation. Aging of the culture adversely affect the production level. Vigorous stirring may damage the cells.
  • 7. WHY DO PLANTS PRODUCE 2°- METABOLITES? For protecting themselves from infections by producing some antimicrobial compounds called phytoalexins. It is believed that production of 2°-metabolites is linked to the induction of morphological differentiation. It is often seen that some of differentiated tissues get specialized to produce specific 2°-metabolites, and in comparison to in vitro cultures, which are the masses of undifferentiated cells, produces higher level of 2°- metabolites.
  • 8. APPLICATIONS OF 2°- METABOLITES: Chemically they may be alkaloids, terpenoids, glycosides etc. Pharmaceuticals, flavors, perfumes, agrochemicals, insecticides and raw materials for industries. Shikonine-A dye from Lithospermum erythrorhizon. Production of 2°-metabolites is a multibillion industry. One kg of vincristine and vinblastine cost
  • 9. Name of the 2°-Metabolites Pharmaceutical Uses Codenine Analgesics Quinine Anti-malarial Atropine Muscle relaxants Digoxin Treatment of cardiovascular disorders Reserpine Hypotensives Jasmine Perfumes Pyrithrins Insecticides Stevioside Food sweeteners Vincristine Anticancer agents
  • 10. STRATEGIES FOR PRODUCTION OF 2°-METABOLITES IN VITRO
  • 11. Selection of cell lines for high yield of secondary metabolites. Large scale cultivation of plant cells Medium composition and effect of nutrients Elicitor induced production of sec. met. Effect of envt. Factors. Biotransformation using plant cell culture. Secondary metabolites release and analysis.
  • 12. I. SELECTION OF CELL LINES FOR HIGH YIELD: Separation of producer cells from the non- producer ones. Euphorbia mili – anthocyanin- Yamada and Fujita (1973) Shikonin-
  • 13.
  • 14. Not all cell types produce the desired metabolite Within a specific cultivar of Catharanthus roseus, 62% of the clones produced the desired metabolite whereas in another only 0.3% produced the metabolite
  • 15. Culture conditions must be optimized e.g. concentrations of sugar, hormones, and vitamins light temperature
  • 16.
  • 17. Metabolite production is frequently higher in cell cultures Berberine production from Coptis japonica is about 5% of dry weight after 5 years of root growth, which equals 0.17 mg/g per week. Whereas in selected cell lines it can be 13.2% of the dry weight in cell culture after 3 weeks, which is about 44 mg/g/week or about 250 times higher
  • 18.
  • 19. Many secondary metabolites are produced in roots Scientists have developed a form of root culture using Agrobacterium rhizogenes, the cause of hairy root disease. (Show Fig 14.3) Cells transformed with some of the bacteria’s DNA, causes the cells to be more sensitive to the hormones they produce. The cells form into roots. These roots grow very fast and produce the secondary metabolites that ordinary roots produce.
  • 20. Root cultures are often better than cell cultures Roots often secrete the metabolites into the surrounding medium, making it easy for collection. Charcoal can be added to the medium, the metabolites are absorbed by the charcoal, and this stimulates even higher production of the metabolite.
  • 21. For optimal production of secondary metabolites a two medium approach is desirable. First medium: reqd for good growth of cells. Second medium: refer to as production medium promotes secondary metabolites production.
  • 22. Effect of precursors Addition of precursors to the medium enhances product formation. Eg: ornithine, phenylalanine, tyrosine and sodium phenylpyruvate, precursors typtamine and secologanin increase ajmalicine production in C.roseus culture.
  • 23. Production- very low, demand- not met… Effort : for product formation at molecular level, and exploit the ways for increased production. Elicitors are the compounds of biological origin which stimulate the production of secondary metabolites, and the phenomenon is called ELICITATION.
  • 24. ELICITORS ENDOGENOUS EXOGENOUS ABIOTICBIOTIC All elicitors of biological origin Physical agent: heat, cold, UV, osmotic pressure Chemical agent: antibiotics, fungicide, etc.. Produced by microbes. Eg: chitin, chitosan, glucans. Within plant cell: pectin, pectic acid, cellulose, etc METHODOLOGY FOR ELICITATION a) Selection of microorganisms b) Co-culture
  • 25. Effect of light Effect of incubation temp Effect of pH Aeration of culture
  • 26. Conversion of one chemical into another by using biological system as biocatalyst is regarded as biotransformation or bioconversion. Conversion of some less imp substances to valuable medicinal or commercially important products. Bioconversion may involve many reaction eg: hydroxylation, reduction, glycosylation.
  • 27. Good eg is : large scale production of cardiovascular drug digoxin from digitoxin by Digitalis lanata. Cell culture Digitalis purpurea or Stevia rebaudiana can convert steviol into steviobiocide and steviocide which are 100 times sweeter than cane sugar,
  • 28. For secondary metabolites stored in vacuoles of cells, two membranes have to be disrupted. Permeabilizing agents such as DMSO can be used for the release of products. Separation and purification is costly, so two approach are made: a) Production of sec. met. Should be high as possible b) Formation of side product which interfere must be minimal.
  • 29.
  • 30. Kinds of Secondary Metabolites alkaloids phenolics (including polyphenols and tannins) terpenoids
  • 31.
  • 32. Precursors can be fed to either cell culture or roots to produce the metabolite in question. In addition, cells can be genetically engineered to over-produce the metabolite, but this may be more difficult with pathways that have many enzymes.
  • 33. Some secondary metabolites produced in cell and root culture L-DOPA: a precursor of catecholamines, an important neurotransmitter used in the treatment of Parkinson’s disease Shikonin: used as an anti-bacterial and anti- ulcer agent Anthraquinone: used for dyes and medicinal purposes
  • 34. Opiate alkaloids: particularly codeine and morphine for medical purposes Berberine: an alkaloid with medicinal uses for cholera and bacterial dysenterry Valepotriates: used as a sedative Ginsenosides: for medicinal purposes
  • 35. Rosmarinic acid: for antiviral, suppression of endotoxin shock and other medicinal purposes Quinine: for malaria Cardenolides or Cardioactive glycosides: for treatment of heart disease
  • 36. Taxol: an example Taxol is a unique anticancer drug from the bark of the Pacific Yew (Taxus breviola)
  • 37. Pacific Yew Facts Pacific Yew was considered a trash tree by foresters The tree is slow growing, taking about 50 years to mature It grows best in the understory of other trees, not doing well in direct sunlight
  • 38. Taxol Facts Very effective treatment against ovarian cancer, breast cancer, melanoma, and colon cancer Stops cell division, thus blocking cancer. It does this by interfering with microtubule function. Microtubules are responsible for pulling apart the sets of chromosomes in mitosis.
  • 39. Taxol Needs It is estimated that 250 kg of pure Taxol are needed to treat cancer in the USA. This would require the bark of about 360,000 trees per year! Obviously Taxol woud be very expensive by this method (approximately $200,000 to $300,000 per kg).
  • 40. Taxol is a very good target for biotechnology a) tissue culture of bark cells b) fungus produces taxol c) alternative species d) genetic engineering e) chemical synthesis
  • 41. a) tissue culture of bark cells Many cells from different bark tissues from different trees were screened. There are at least 25 fold differences in production. It was found to be secreted into the medium thus facilitating collection. So far 1 to 3 mg of taxol are produced per liter of cell culture. This is equivalent to about 25 g of bark.
  • 42. b) fungus produces taxol It was found that a fungus that colonizes yew trees also produces taxol Fungal culture technology which is better developed than plant cell culture technology could be an important source for taxol production
  • 43. c) alternative species Some researchers found that the European Yew (Taxus baccata) produces a precursor to taxol. This precursor can then be converted to an analog of taxol in the laboratory. The precursor is used for chemical synthesis of taxol.
  • 44. d) genetic engineering Other scientists are trying to identify and clone the genes which produce taxol This will enable them to scale up production in cell culture
  • 45. e) Chemical synthesis Until 1994, chemical synthesis was formidable 3 different ways to synthesize taxol are now known Some take up to 13 steps Cost per patient still expensive; about $20,000
  • 46. This is because it usually takes 10 years of research to produce a product. This requires that a product sell for at least $400 per kg to make it economically worthwhile.
  • 47.
  • 48. Many of these Third World countries may lose market share to superior, more efficient production of secondary metabolites in industrial countries. Is this right? Is it fair? Are third world countries capable of competing? What should they do?