It would be helpful to Bachelor as well as Masters students Dr. S. Prashant

1. Strategies to enhance production
of Secondary Metabolites

2. Strategies to enhance production of
Secondary Metabolites
• Screening of high-growth cell lines to produce
secondary metabolite of interest – Cell line selection
• Use of elicitors to enhance productivity in a short
period of time
• Adsorption of metabolites to overcome feed back
inhibition
• Scale-up of cell cultures in suitable bioreactors
• Immobilization of cells to enhance yields of extra-
cellular metabolites and to facilitate biotransformation
• Use of Organ Cultures – Hairy Root cultures
• Permeabilization of secondary metabolites to recovery
or downstream processing

4.  Plant cell culture is a genetically heterogeneous system.
In addition, epigenetic changes cause genetic instability leading to
product accumulation only in some population of cells.
The overall production of secondary metabolites in a cell culture depends
on the rate of accumulation within the productive cells.
By cell culture system only low yields of desired secondary metabolites
are obtained.
The reason for low production in culture may be due to:
 Competition between primary and secondary pathways for common
intermediates
 Low levels of expression of key enzymes at rate limiting steps in a
pathway
Screening is a passive technique by which a great number of cells alone
analyzed for a certain traits and those showing the desired features are
cultivated and screened.
Selection process is an active process, which deliberately favor only the
survival of the desired variants while wild type cells are killed.
1. Selection of cell lines with high productivity

5. 1. Selection of cell lines with high productivity
• Identify a genotype with high levels of desired secondary
metabolite
• Callus from such a genotype serves as the source of
desirable cell lines
• Screening for Selection of cell lines with high productivity
• Screening for high yielding lines for coloured compounds
like shikonin, berberine and betain is easy – most coloured
cell clumps can be isolated and cultured separately
• Cells with highly fluorescent metabolites/compounds can
be detected using UV microscope, Flowcytometry
• HPLC and RIA are also used to screen high yielding cell lines
• Selected cell lines should be stable, repeated selection
process would ensure isolation of true variant cell line

6. • Two methods followed:
• 1. Single cell cloning - Single cells are taken
from suspension cultures and grown into
population.
• 2. Cell aggregate cloning- Cell aggregates are
separated from suspension cultures and
plated on solid medium and cultured.
Selection of cell lines with high productivity

7. 1. Selection of cell lines with high productivity
Cell line selection in Panax sikkimensis root callus cultures

8. 2. Use of elicitors to enhance productivity
of secondary metabolites
• In nature plants produce many compounds in
response to biotic and abiotic stresses
• Stress due to pathogen infection or exposure
adverse physical conditions enhance the
production of secondary metabolites.
• The phenomenon of accumulation of secondary
metabolites in response to biotic or abiotic stress
conditions is known as Elicitation.
• The compounds or agents which induce
elicitation are called Elicitors.

9. Elicitors
• Elicitors can be either biotic or non-biotic in nature.
• Biotic elicitors:
• Fungal extracts or toxins
• Bacterial extracts or toxins
• Culture filterates
• Compounds obtained from microbial cell walls – polysaccharids,
glycoproteins, chitosons etc.
• Yeast extracts
• Abiotic elicitors: Physical and chemical agents
• UV radiation
• Exposure cold or heat
• Ethylene
• Fungicides
• Antibiotics
• Heavy metal salts
• High Salt concentration

10. Mechanism of Elicitation
• Elicitors bind to the receptors present on the
plasmamembrane and trigger signal
transduction and activating enzymes involved
in production of secondary metabolites.
• Elicitor induced products are continuously
released in to medium.
• This allows continuous production of
secondary metabolites.

11. Factors affecting Elicitation
• Sensitivity of the cell cultures
• Specificity of elicitors
• Concentration of elicitors
• Timing and duration of elicitor exposure to
culture medium
• Most of the cells respond to elicitors only
during growth phase

12. 3. Immobilization of Cells
• In suspension cultures plant cells grow faster and
produce less secondary metabolite.
• Cell aggregates have better ability to produce &
accumulate secondary metabolites than free floating
suspensions.
• It is essential to anchor the plant cells on some support
or entrap the cell in a matrix such as gel, agar, alginate,
polyacrylamide etc.
• Such a process of anchoring plant cell is known as
Immobilization.
• Brodelius et al. (1979) first reported the immobilization
of C. roseus and Daucus carota cells by entrapping
them in alginate beeds.

13. Steps in Immobilization Process
• Preparing a support – substantially uniform
and porous matrix of inorganic material
• Introducing culture of viable plant cells into
the pores of matrix
• Entrapping the plant cells by coating the
matrix with a solution or colloidal suspension
not interfering with the cell viability
• Immobilizing the entrapped cells within the
matrix with a reactive gas .

15. IMMOBILIZATION TECHNIQUES

16. IMMOBILIZED CELL BIOREACTOR

17. Permeabilization of
secondary metabolites
• Many secondary metabolites are hydrophilic, so they
are primarily stored in the cell vacuole.
• Hydrophobic molecules are often stored in the cell
membrane, vesicles, dead cells and extracellular sites,
such as the cell wall.
• By manipulating the permeability of cell membrane,
they can be secreted out to the media.
• Permeabilisation can be achieved by electric pulse, UV,
pressure, sonication, heat, etc.
• To maintain cell viability and allow for further use of
biomass, cells can be permeabilized using pH shock or
chemical treatments like DMSO.

18. Production of secondary metabolites using
Hairy Roots