Secondary metabolites, synthesis process and importance

1. Production of Secondary Metabolites
Bhagyashree M. Shelar
Dr. D. Y. Patil Biotechnology and Bioinformatics Institute.
MIT 721
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2. METHODS FOR PRODUCTION OF SECONDARY
METABOLITE’S
1)Cell Cloning of Auxotrophic cells
2)By immobilisation of plant cell
3)Two –phase system culture
4)Hairy root culture
5)Bio transformation of plant cells
6)Using genetically engineered plant cell cultures
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Hairy Root Culture Bioreactor Immobilisation of
plant cell

3. 1)CELL CLONING OF AUXOTROPHIC CELLS
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Identify high-yielding
plant
Explant culture
Establish callus culture Establish cell suspension
Re-plate and select for high product biosynthesis thg. media
manipulation and environmental stress
Scale up suspension culture
Bioreactor production

4. TECHNIQUES OF SELECTING CELL LINES FOR
HIGH YIELDS OF SECONDARY METABOLITES
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1.Cell cloning
2.Visual or Chemical Analysis
3.Selection for resistance
4.Auxotrophy

5. TECHNIQUES OF SELECTING CELL LINES FOR
HIGH YIELDS OF SECONDARY METABOLITES
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1.Cell cloning
 Tool to explore heterogeneity in cell
culture and selecting high yielding
cell lines.
 Simplest procedure is to take single
cells from suspension culture and
grow them individually on suitable
medium and then screen each
population for alter phenotype
OR
 Alternatively suspension culture
screened for phyto-chemical
differences by passing through sieves
of definite mesh size followed by
density gradient centrifugation
 e.g. Daucus carota for anthocyanin ,
Lithospermium erythrorhion for
shikonin, Nicotiana tobaccum for
nicotine etc.
2.Visual or Chemical Analysis
 For identification of high yielding cells
 Visual analysis uses microscopic
estimation of compounds which are
coloured.
 This is non destructive useful in cloning
by identifying cells or callus with high
concentration of pigments.
 E.g.β-carotene in carrot , shikonin in
Lithospermium cultures.
OR
 Chemical analysis utilise analytical
techniques which are rapid,
inexpensive, specific and sensitive
e.g. Flow cytometry , radioimmunoassay
 E.g.Cathranthus for serpentine and
ajmalicine

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http://phiv.cirad.fr/download/poster.pdf
Multiphoton microscopy e.g. Epi-fluorescence microscopy

7. TECHNIQUES OF SELECTING CELL LINES FOR
HIGH YIELDS OF SECONDARY METABOLITES
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3.Selection for resistance
 Selection is for resistance(mutant cell
lines) to toxic compounds
 Such mutant selected by their ability
to grow in presence of inhibitor
 Cells which overproduce primary
metabolite dilute toxic effect of their
analogue
 E.g. Cell lines resistance to
tryptophan analogue 5-
methyltryptophan overproduce
tryptophan this is due to mutation in
feedback mechanism , Catharanthus
roseus cell resistance to 4MT
resulted in accumulation of ajmalicine
4.Auxotrophy
 is the inability of an organism to
synthesize a particular organic
compound required for its growth.
 Auxotrophs selection useful for
increase production of secondary
metabolites since block in
necessary pathways results in build
up of intermediates which are
precursor of secondary compounds.
 Disadvantage is can be done in few
cases and selection done only in
haploid cells since auxotroph’s are
mutants of recessive traits which is
not a easy task.

8. 2)BY IMMOBILISATION OF PLANT CELL
Methods of immobilisation is based on gel or
membrane entrapment
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Entrapment in
Gels
Entrapment in
Hollow-Fibre
Membrane
Bioreactor
configurations used
with immobilised
cells
Entrapment
in Nets or
Foam

9. IMMOBILISATION OF PLANT CELL
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Entrapment in Gels
 Cells or protoplast entrapped in
alginate or carrageenin or agar
or agarose etc. Or combination
of gel and allow them to
polymerise around them.
 E.g. C. Roseus, Digitalis
lanata in calcium alginate
Entrapment in Nets or Foam
 Cells growing in medium can be
readily immobilised in blocks of
polyurethane foam which is used in
1cm3.
 blocks of gel immersed in cell
suspension and flask agitated on rotary
shaker initially free cell and cell
aggregates washed in-out of blocks but
soon it become trapped deep in matrix
 After two weeks or when entire block
has filed with cells the cubes
transferred to low growth medium
which support metabolic activity but
nit active cell division
 E.g. Immobilised cells of capsicum
produce more capsaicin than in free
suspension under similar growth
condition

10. IMMOBILISATION OF PLANT CELL
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Entrapment in Hollow-
Fibre Membrane
 Cells can be entrapped in spaces
between the fibre membranes
these spaces are permeable to
nutrients and precursor of
secondary metabolite's
 Expensive but mechanically
stable and offer better control of
fluid and flow distribution
 Hollow fibre cartridges used for
immobilisation of Daucus carota
Bioreactor configurations
used with immobilised cells
 Bioreactor used are fluidised-bed and
fixed –bed categories
 In fluidised system immobilised cells
agitated by flow of air or by pumping
medium through bioreactor contrarily
in fixed bed bioreactor immobilised
cells held at stationary and perfuse
with aerated liquid medium at slow
rate
 In both type cells are protected from
shear stress though fluidised-bed
reactor are more effective

11. 3)HAIRY ROOT CULTURE
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Screening on productivity
Plant Root
Agrobaterium rizogenes
Hairy Root Lines
Production in
Bioreactor
Entrapment in Nets or
Foam

12. APPLICATIONS
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References
1. Introduction to Plant Tissue Culture M.K. Razdhan
2. Early Approach to Plant Secondary Metabolite by Biyani
3. http://link.springer.com/book/10.1007%2F978-1-4615-
4913-0
4. http://pctre.com/plant-metabolites.html
5. http://www.northeastern.edu/lee-parsons/research/
6. https://www.researchgate.net/publication/236119384_Bioe
ncapsulation_of_microbial_inoculants_for_better_soil-
plant_fertilization_A_review

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