In this presentation you can familiar with: Primary metabolite vs secondary metabolite Importance and function of secondary metabolite Approaches for increasing secondary metabolite production in plant tissue culture

1. Current approaches toward production of
secondary plant metabolites
By: Shahnam Azizi
Department of Biotechnology,
Faculty of Agriculture, Azarbaijan
Shahid madani University, Tabriz,
Iran
Autumn 2017

2. outline
Primary metabolite vs secondary metabolite
Importance and function of secondary
metabolite
Approaches for increasing secondary metabolite
production in plant tissue culture
2

3.  For million years, humankind is completely dependent on plants
as source of food and shelter
 plants are a valuable source of a wide range of metabolites Used
as pharmaceuticals, agrochemicals, flavours, fragrances,
colours, bio-pesticides and food additives.
 Nearly 70% - 80% of world population depends upon herbal
drugs.(WHO)
3
Plants have a major role in human lifePlants have a major role in human life

4. 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.
4
vsvs
Amino acid

5. There are three potential pathways for primary metabolism
Embden Meyerhof-Parnas Pathway (EMP)
Entner-Dourdorof pathway
hexose monophosphate (HMP) pathway
5

6. Feature and function of secondary metabolite
 They are biosynthetically derived from primary metabolites
 Produces product that aid in the growth and development of plant
but not required for the plant to survive.
 They facilitate the primary metabolism in plant
 They are the major component of plant defense mechanism
against herbivores, pest and pathogen
6

7. Features and functions of secondary
metabolite
Attract pollinators or seed dispersal agents
Important for abiotic stresses
Medicine
Industrial additives
7

8. They are classified on the basis of
1) chemical structure
2) chemical composition
3) solubility in various solvents
4) pathways by which they are synthesized
8
Classification of secondary metabolite

9. 1)The terpenes- made from mevalonic acid, composed almost
entirely of carbon and hygrogen
2) phenolics – made from simple sugars, containing benzene
rings, hydrogen and oxygen
3) nitrogen-containing compounds – extremely diverse, may also
contain sulphur
9
A simple classification of secondary metabolites includes three
main groups

10. 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
10

11. 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 alkaliods 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
11
Role of Endophytes in In vitro Production of
Secondary Metabolites

12. 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
12

13. 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 13
Major Advantages

14. 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
Limitations/Disadvantages
14

15. Strategies for increasing secondary
metabolites in vessel culture
optimizing the cultural conditions
 selecting high-producing strains
employing precursor feeding
transformation methods
immobilization techniques
15

16. Hairy root cultures as a source of secondary metabolites
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).
16
hairy root phenotype is characterized by

17. 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
17

18. 18

19. 19
Physalis alkekengi secondary metabolite
Induction of hairy roots by various strains of Agrobacterium
rhizogenes in Physalis alkekengi from hypocotyl and cotyledon
explants
Physalis alkekengi

20. 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. 20

21. 21
Elicitors Classification Based on their Nature
Poornananda M. Naik and Jameel M. Al–Khayri (2016)

22. 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 22

23. 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)
23Rosmarinic acid

24. 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
24

25. 25
IMMOBILIZATION OF PLANT CELLS
Alginate
Polyacrylamide
Agar and agarose
Polyuretane
Adsorption
Covalent linkage
Entrapment

26. 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
26

27. 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
27
Metabolic Engineering
various strategies to improve secondary metabolite
by M.En

28. 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
28

29. References
 HU, Z-B., DU, M. (2006): Hairy root and its application in plant genetic
engineering. Journal of Integrative Plant Biology, 48, (2): 121-127.
• GIRI, A., NARASU, M., (2000): Transgenic Hairy Roots: Recent Trends
and Application. Biotechnology Advances, 18: 1-22.
• Poornananda M. Naik and Jameel M. Al–Khayri (2016). Abiotic and Biotic
Elicitors–Role in Secondary Metabolites Production through In Vitro
Culture of Medicinal Plants, Abiotic and Biotic Stress in Plants - Recent
Advances and Future Perspectives, Arun K. Shanker and Chitra Shanker
(Ed.), InTech, DOI: 10.5772/61442.
• Ellis BE, Towers GH. Biogenesis of rosmarinic acid in Mentha.
Biochemical journal. 1970 Jun 1;118(2):291-7.
• Romagnoli, L. G. and Knorr, D. 1988. Effects of ferulic acid treatment on
growth and flavour development of cultured Vanilla planifolia cells. Food
Biotechnology, 2: 93-104.
29