Current approaches toward production of secondary plant metabolites

1. CURRENT APPROACHES TOWARD
PRODUCTION OF SECONDARY PLANT
METABOLITES
By : Aswin M B
Biochemistry and molecular
biology,
Central university of Kerala
BBM052007

2. OUTLINE
❑Primary metabolite vs secondary metabolite.
❑Importance and functionof secondary metabolite.
❑Approaches for increasing secondary metabolite production in plant
tissue culture.
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3. PRIMARY METABOLITE VS SECONDARY
METABOLITE
• Primary metabolites are compounds that are directly involved in the
growth and development of a plants they are includes
1) Carbohydrates 2) Proteins 3) Lipids 4) Nucleic acids 5) Hormones
• Secondary metabolites that are not essential for growth and
development of an organism.
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4. Major Primary metabolite pathway
Embden Meyerhof-Parnas Pathway (EMP)
Entner-Dourdorof pathway
Hexose monophosphate (HMP) pathway
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5. FEATURE AND FUNCTION OF SECONDARY
METABOLITE
➢They are biosynthetically derived from primary metabolites.
➢This Produces product that aid in the plant to survive but not required for
the growth and development of plant.
➢ They are the major component of plant defense mechanism against
herbivores, pest and pathogen.
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6. ROLE OF SECONDARY METABOLITES
 For protect the plant from invading pathogens.
 Helps to improve the shelf life of plant.
 Helps to keep the plant healthy.
 Decreases the infection from some diseases and pests also.
 Useful in preparation of perfumes, or aromatic substances.
 It has virtually important in attracting the animals for pollination.
 Sometimes for else were for seed dispersal.
 Decreases the use of insecticides, pesticides, and fungicides also.
 It improves the immunity of plant.
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7. CLASSIFICATION OF SECONDARY METABOLITE
The classification are on the basis of –
1) chemical structure
2) chemical composition
3) solubility in various solvents
4) pathways by which they are synthesized
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8. ROLE OF ENDOPHYTES IN IN VITRO PRODUCTION OF
SECONDARY METABOLITES
❑ There are three schools of thought on the origins of secondary metabolism
in plants
I. Both plants and endophytic microbes coevolved with pathways to produce
these natural products.
II.An ancient horizontal gene transfer took place between plants and microbes.
III.Either plants or endophytic fungi produce these secondary metabolites and
transfer them to the other symbiont.
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9. ROLE OF ENDOPHYTES IN IN VITRO
PRODUCTION OF SECONDARY METABOLITES
The symbiotic association and effects of plants and endophytes on each other during
the production of other important pharmacological bioactive natural products
➢Claviceps sp: producing ergot alkaloids was a potential source of useful secondary
metabolite (Clay, 1988).
➢Gibberella fujikouri an important endophytic fungus in rice (Oryza Sativa) was the
source of the phytohormone Gibberellin (Steierle et al.,1993)
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11. THE ADVANTAGES AND LIMITATIONS OF IN VITRO
CULTURE IN PRODUCTION OF SECONDARY METABOLITE
ARE LISTED
• Major Advantages
1.Compounds can be produced under controlled conditions
as per market demands.
2.Culture systems are Independent of environmental factors,
seasonal variations, pest and microbial diseases and geographical
constraints.
3.Cell growth can be controlled to facilitate improved product
formation.
4.The quality of the product will be consistent as it is produced
by a specific cell line.
5. Recovery of the product will be easy. 11

12. 6)Plant cultures are particularly useful in case of plants which are
difficult or expensive to be grown in the fields
7)Mutant cell lines can be developed for the production of novel
compounds of commercial importance, which are not normally found in
plants.
8)Biotransformation reactions (converting specific substrates to valuable
products) can be carried out with certain cultured cells.
9)The production control is not at the mercy of political interference.
10)The production time is less and labour costs are minimal
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13. LIMITATIONS/DISADVANTAGES
1) In general, in vitro production of secondary metabolites is lower when
compared to intact plants.
2) Many a times, secondary metabolites are formed in differentiated
tissues/organs. In such a case, culture cells which are non-differentiated
can produce little.
3) Cultured cells are genetically unstable and may undergo mutation
4) The production of secondary metabolite may be drastically reduced,
as the culture ages
5) Vigorous stirring is necessary to prevent aggregation of cultured
cells. This may often damage the cells.
6) Strict aseptic conditions have to be maintained during culture technique:
Any infection to the culture adversely affects product formation 13

14. ➢optimizing the cultural conditions
➢selecting high-producing strains
➢employing precursor feeding
➢transformation methods
➢immobilization techniques
Strategies for increasing secondary
metabolites in vessel culture
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15. IMMOBILIZATION OF PLANT CELLS
➢It is a technique, which confines the cells to a defined region in a space while retaining
their catalytic activity and prevents its entry into the mobile phase, which carries the
substrate and product.
➢Immobilization of plant cells would be one method of increasing productivity and
hence reducing the costs.
➢Immobilization of plant cells, protoplast or embryos is achieved by binding these
materials onto or within a solid support.
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16. IMMOBILIZATION OF PLANTCELLS
Alginate
Polyacrylamide
Agar and agarose
Polyuretane
❑Adsorption
❑Covalent linkage
❑Entrapment 16

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18. HAIRY ROOT CULTURES AS A SOURCE OF SECONDARY
METABOLITES
• hairy root phenotype is characterized by
➢Hairy roots grow rapidly.
➢show plagiotropic growth (highly branched ).
➢Propagate in phytohormone-free medium (HU AND DU,
2006).
➢Hairy root cultures produce secondary metabolites over
successive generations without losing genetic or biosynthetic
stability (GIRI VE NARASU, 2000).
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19. IMPORTANT FACTORS IN ESTABLISHMENT OF A HAIRY ROOT
CULTURE SYSTEM
➢Bacterial strain of A. rhizogenes.
➢An appropriate explant.
➢A proper antibiotic to eliminate redundant bacteria after
cocultivation.
➢A suitable culture medium.
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21. Advantages of hairy root culture
•Harvesting roots for extracting secondary metabolites can cause destruction to whole plants. Therefore,
interest in producing secondary metabolites by developing hairy root culture has been raised.
•Hairy root culture potentially grows faster without needing an external supply of auxins. In certain cases,
they do not need incubation under light.
•Due to their high genetic stability all hairy root cultures are stable in metabolite production.
•Yield in hairy root cultures can be altered by optimizing various factors such as carbon source and its
concentration, ionic concentration of the medium, pH of the medium, light, temperature, and inoculum.
•In addition utilization of techniques like precursor feeding, cell immobilization, elicitation, and
biotransformation of hairy root culture can improve secondary metabolite production.
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22. 22

23. INDUCTION OF HAIRY ROOTS BY VARIOUS STRAINS OF
AGROBACTERIUM RHIZOGENES IN PHYSALIS ALKEKENGI FROM
HYPOCOTYLAND COTYLEDON EXPLANTS
Physalis alkekengi
Secondary metabolites
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24. ELICITATION OF IN VITRO PRODUCTS
➢ Plants and/or plant cells in vessel culture 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.
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25. Elicitors Classification Based on their Nature
Poornananda M. Naik and Jameel M. Al–
Khayri (2016)
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26. PRECURSOR FEEDING
➢ precursor feeding has been an obvious and popular approach to increase secondary
metabolites production in plant cell cultures.
➢ Precursor feeding: exogenous supply of a biosynthetic precursor to culture medium
may also increase the yield of the desired product.
➢ This approach is useful when the precursors are inexpensive.
➢ With the basis of the knowledge on biosynthetic pathways, several organic compounds have
been added to the culture medium in order to enhancing the synthesis of secondary
metabolites.
➢ Precursor feeding is based on the idea that any compound, which is an intermediate, in or
at the beginning of a secondary metabolite biosynthetic route, stands a good chance of
increasing the yield of the final product.
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27. SOME EXAMPLES FOR PRECURSOR FEEDING
✓amino acids have been added to cell suspension culture media for production
of tropane alkaloids, indole alkaloids etc
✓Addition of phenylalanine to Salvia officinalis cell suspension cultures
stimulated the production of rosmarinic acid (Ellis and Towers 1970)
✓Feeding ferulic acid to cultures of Vanilla planifolia resulted in an increase
in vanillin accumulation (Romagnoli and Knorr 1998)
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28. METABOLIC ENGINEERING AND PRODUCTION OF SECONDARY
METABOLITES
❑ In many cases production of secondary metabolite is too low for
commercialization, metabolic engineering can provide various strategies to
improve productivity.
Metabolic engineering
Metabolic engineering is the alteration of cellular activities by the
manipulation of enzymatic, transport, and regulatory functions of the cell by
using recombinant DNA technology.
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29. VARIOUS STRATEGIES TO IMPROVE SECONDARY METABOLITE
BY METABOLIC ENGINEERING
a) Increasing the number of producing cells.
b) Increasing the carbon flux through a biosynthetic pathway by overexpression
of genes.
c) Codify for rate-limiting enzymes or blocking the mechanism of
feedback inhibition and competitive pathways.
d) Decrease catabolism.
various strategies to improve secondary metabolite by M.En
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30. BIOREACTORS SCALING UP OF PRODUCTION OF SECONDARY
METABOLITES
➢ Better control of the culture conditions
➢ Optimal supply of nutrients and growth regulators
➢ Renewal of the culture atmosphere
➢ Changing the medium during the culture period according to the developmental stage
➢ Filtration of the medium for exudates
➢ Contamination control
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31. REFERENCES
• Hussain MS, Fareed S, Ansari S, Rahman M, Ahmad IZ, M Saeed. Current approaches toward production of
secondary plant metabolites. J Pharm Bio all Sci 2012;4:10-20.
• HU, Z-B., DU, M. (2006): Hairy root and its application in plant genetic engineering. Journal of Integrative Plant
Biology, 48, (2): 121-127.
• U.Satyanarayana. Biotechnology. 2017;12:508-516.
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