IN VITRO SECONDARY METABOLITE PRODUCTION

In vitro secondary metabolites

• In vitro production of secondary metabolites involves culturing plant
cells, tissues, or organs in a controlled environment (like a laboratory)
to synthesize valuable plant-derived compounds.
• This technique offers an alternative to traditional methods of sourcing
these compounds from plants in their natural environment, which can
be limited by factors like geographic location, seasonal availability,
and ethical concerns.
• By manipulating culture conditions, elicitors, and other factors,
scientists can enhance the production of desired secondary
metabolites.
Chemical structures of some bioactive plant secondary metabolites used in medicine
Methods for the production of bioactive secondary metabolites
through plant in vitro culture.
• Establishment of in vitro cultures after surface sterilization of plant material.
• Callus formation can also be induced on material from organ cultures. Reversely,
organs like roots or shoots can be regenerated from callus.
• Hairy roots are obtained by infection of sterilized donor plant, or in vitro cultivated
material, with Agrobacterium rhizogenes.
• Multiplication of primary callus/organs/roots, first selection, and establishment of
liquid cultures.
• Selection of high yielding lines and optimization of culture conditions (nutrient
medium composition, inoculum density, temperature, light, agitation and aeration).
• Strategies like elicitation, precursor feeding, or immobilization are pursued to
further improve productivity.
• The bioreactor design will depend on the culture type: stirred tanks, but also airlift
and bubble column reactors for cell suspensions; mist or spray reactors and
temporary immersion systems for organ cultures (including hairy roots)
Strategies for optimizing product yield
• The production of secondary metabolites by in vitro cultures usually
occurs in a two-step process, biomass accumulation and secondary
metabolites synthesis, in which both steps need to be optimized
independently.
1. Selection of High-Yielding Cell Lines
2. Optimization of culture conditions
3. Elicitation
4. Nutrient and precursor feeding
5. Immobilization of cultures
6. Permeabilization of plant cell membranes
7. Hairy root culture
8. Biotransformation
Selection of High-Yielding Cell Lines
• Not all cells in a culture produce secondary metabolites at the same level. By
screening and selecting variants that show enhanced production capacity,
stable high-yielding cell lines can be developed. This is often the first and most
critical step in optimizing metabolite production.
• Basis of genetic variation:
• Somaclonal variation - Genetic/epigenetic changes during repeated subculture.
• Epigenetic modifications - DNA methylation and chromatin remodeling can alter metabolite
pathway gene expression
• Mutations - Spontaneous or induced by mutagens (UV, EMS).
• Metabolic heterogeneity - Natural differences among cells in enzyme levels/pathway flux.
• Selection Strategies : Screening for Metabolite Content, Visible Phenotype Selection,
Selection Using Elicitors/Stress
Examples:
• Shikonin (from Lithospermum erythrorhizon): High-yielding purple callus lines selected and commercialized in
bioreactors.
• Taxol (from Taxus spp.): Cell suspension lines with >20× higher yields identified after multiple screening
cycles.
• Anthocyanin (from carrot cell culture): Pigmented high-producing lines stabilized for industrial extraction.
[Link]
Optimization of culture conditions
• There are several factors that can be optimized to improve the growth and
metabolites productivity of the in vitro cultures.
• Among them, the following can be appointed: the culture medium composition,
the medium pH, the inoculum density, the culture medium environment (e.g.,
temperature, light density and quality, etc.), the agitation and aeration.
• The culture medium strongly affects the biomass and metabolites productivity.
• It must be selected according to the physiological requirements of the plant
species, and there are several parameters that can be optimized, namely
nutrients composition, salt strength, nitrate and phosphate levels, plant growth
regulators type and concentration, carbon source, etc.
• For instance, carbon source plays significant roles in the signal transduction
systems through regulating gene expression and developmental processes.
➢ Higher sucrose - anthocyanin induction; phosphate limitation - alkaloid stimulation.
➢ Auxins/cytokinins influence differentiation and metabolite pathways - 2,4-D + kinetin combinations for
induction of alkaloids in callus.
Elicitation
• It is the process of in vitro exposing plants to trace levels of elicitors in the
growth media in order to promote metabolite production.
• An elicitor is a substance that stimulates plant defense and consequently
boosts secondary metabolite biosynthesis to protect the cell and the plant
as a whole. These compounds can be abiotic (jasmonic acid, heavy
metals, UV light, etc.) or biotic (chitosan, yeast extract, etc.).
• Ultimately, we "trick" the plant with stress signals to activate its defensive
system. In fact, elicitation is one of the most effective ways for altering the
quantitative and qualitative production of secondary metabolites.
• Elicitors have the ability to control an array of cellular activities at the
biochemical and molecular level since they induce the upregulation of
genes.
Selected examples of elicitation treatments applied in cultures of Lamiaceae species to increase the
production of rosmarinic acid.
Immobilization of cultures
• Immobilization of plant cells is another strategy used to overcome
problems of low shear resistance and cell aggregation.
• This procedure can be done by several methods, and the most widely used
are surface immobilization or gel entrapment.
• In this technique, the cells are entrapped in a speciic gel or a combination
of gels.
• Examples of matrices used are calcium alginate (the most used), agarose,
gelatin, carrageenan, or polyacrylamide.
• This strategy has several advantages, such as the extension of cells’ viability
in the stationary stage, the simplification of downstream processing, the
high-cell density within small bioreactors reducing the costs and risk of
contamination, an increased product accumulation, the minimization of
fluid viscosity.
Nutrient and precursor feeding
• Nutrient and precursor feeding are also used to improve the yields of secondary
metabolites production.
• Nutrient feeding involves the replenishment of nutrient medium, and in precursor
feeding, plant cell cultures are used to convert precursors into products by utilizing
preexisting enzyme systems.
• Plants employ initiator or intermediary chemicals, known as precursors, as the
foundation for the majority of secondary metabolites (SM). So, the underlying
concept of this technique is that any of these molecules, when added at the start of
biosynthesis, can boost the creation of the final product.
• Precursor feeding is the most cost-efficient technique for stimulating SM
production and is effective in a wide range of plant species.
• Shikimic acid, jasmonic acid, and amino acids
like phenylalanine and tryptophan are all frequent precursors.
• Depending on the physicochemical property of the SM to be generated, they can be
introduced during medium preparation or at intervals of growth periods.
• Example - Feeding tryptophan/tryptamine in C. roseus to enhance
catharanthine.
Permeabilization of plant cell membranes

• The permeabilization of plant cell membranes with chemicals, the use

of electric field stress, and ultrasound techniques are strategies used
to facilitate the removal of secondary metabolites from vacuoles and
membrane systems of the plant cell, facilitating the secretion of
products into the culture medium and thus simplifying the
purification process.
• Use surfactants (Tween, DMSO) or membrane modifiers to facilitate
metabolite release.
• Example - Use surfactants enhances secretion of shikonin into
medium.
Biotransformation
• The cultivated cells have the capacity for biotransformation of
supplied compounds, which are not necessarily natural
intermediaries of plant metabolism, into high-value compounds.
• Use cultured cells to convert simple substrates into complex bioactive
products.
• This can occur through different reactions as hydroxylation, oxidation
of hydroxyl group, reduction of carbonyl group, hydrogenation of
carbon-carbon double bond, glycosyl conjugation, and hydrolysis,
catalyzed by plant enzymes.
• Cells or enzymes contain specific enzymes (oxidases, hydroxylases,
glycosyltransferases, etc.) that can catalyze stereospecific and
regioselective reactions which are difficult to achieve chemically.
• Example: Adding a hydroxyl group (-OH) at a specific carbon in a
complex molecule.
Examples of Biotransformation in Plant Biotechnology

Substrate Biotransformed Product System

Digoxin precursor (digitoxin) Digoxin (cardiac glycoside) Digitalis cell cultures

Steroids (progesterone) Cortisone, hydrocortisone Rhizopus species

Ajmalicine Serpentine Catharanthus roseus cultures

Taxodione Taxol derivatives Taxus cell cultures

Shikonin precursors Hydroxylated pigments Lithospermum cultures

 Hairy root culture
Outlined diagram of Agrobacterium rhizogenes mediated
infection to the plant cell, induction and fully grown hairy root
culture, producing bioactive compounds, and their
enhancement strategies.
Factors affecting the production of secondary metabolites in
tissue culture
• Media Formulation: Composition and types of media used for the cell culture
of plants impact the production of secondary metabolites. For example, the
use of MS (Murashige and Skoog) media in the suspension culture
of Catharanthus roseus increases the production of serpentine metabolite.
The effect of media on metabolite yield varies from one plant species to
another.
• Phosphate: Several studies report that the level of phosphate in tissue
culture media affects the production of secondary metabolites.
• For example, in many plants, it has been observed that increasing the phosphate level
in the media increases cell growth, however, reduces the secondary metabolite yields.
• In Catharanthus roseus, the yield of phenolics and ajmalicine; in Nicotiana tabacum, the
yield of caffeoyl putrescine; and in Peganum harmala the yield of Harman alkaloids has
been found to enhance after reducing the phosphate level in their tissue culture media.
• Carbon Source: Sucrose is one of the essential components of plant
tissue culture media. It has been observed that altering the sugar
concentration in the media or using different carbon sources impacts
the production of secondary metabolites in some plants.
• For example, the use of glucose, instead of sucrose, in the media formulation
of Podophyllum hexandrum, increases the production of podophyllotoxin.
Whereas, to increase rosmarinic acid, an increase in the sucrose
concentration level was found to be an effective approach.
• Nitrogen: Nitrogen is one of the essential components of some tissue
culture media, including MS, LS, or B5 media. It has been observed
that media containing amino acids and proteins have better
secondary metabolite yields. Moreover, the amount of that nitrogen
also impacts the production of the metabolites.
• Agitation: During cell suspension culture, it has been found that the
speed of agitation impacts the cell viability, resulting in impacting the
production of secondary metabolites. In Podophyllum hexandrum, a
speed of 200 rpm was found to be damaging for plant cells, whereas
more viable cells were obtained when speed was in the range of 125- 150
rpm.
• Plant growth hormones: Several studies report that the use of types of
hormones and their concentration in the media affect the plant’s growth
in tissue culture, in addition to the production of secondary metabolites.
• For example, some studies found that 2, 4-D ceased metabolite production,
whereas, the addition of NAA and IAA increases the metabolites synthesis.
• In Catharanthus roseus, auxin alone was able to induce callus production,
however, an increase in secondary metabolite production was only observed after
growing the plant alone with cytokinin.
• Elicitation: It’s a process of inducing effects like pathogen attacks in
plants to induce the expression and functioning of genes that are
responsible for the synthesis of secondary metabolites. Both biotic
and abiotic elicitors are used for this purpose. It includes jasmonic
acid, glucan polymers, glycoproteins, fungal cell materials, UV
irradiation, salts of heavy metals, and many other chemicals.
• It has been found that in plants Rauwolfia species and Eschscholzia
californica, the use of yeast extracts as elicitors increased the production of
secondary metabolites. A similar case was observed with using methyl
jasmonate with other plants.
• Gas composition: In some cases of secondary metabolites production,
it has been observed that the percentage concentration of gasses
inside bioreactors impacts the yield.
• For example, in Panax ginseng, 40% oxygen in the chamber was found to have
the highest yield whereas, increasing the concentration to 50% or lowering it to
20-30% resulted in reduced secondary metabolite production.

• Precursor supplementation: Some studies have found that

supplementing the cell cultures with compounds that are also
produced during secondary metabolite synthesis increases their yield.
This process of the addition of such compounds to cell cultures is
known as precursor feeding.
Applications of SMs by Industry:
Pharmaceuticals:
• A significant portion of drugs are derived from medicinal plants, with secondary
metabolites acting as active ingredients.
• Anticancer agents: Compounds used in cancer treatment.
• Antimicrobial agents: Antibiotics and other compounds used to combat
infections.
• Anti-inflammatory, antihypertensive, and antidepressant agents: Drugs for
various medical conditions.
• Analgesics: Pain-relieving compounds.
 Food Industry
• Flavors and fragrances: Used to improve the sensory qualities of food.
• Coloring agents: Natural pigments for coloring foods.
• Preservatives: Antioxidant and anti-browning agents that extend shelf life.
• Nutraceuticals: Dietary Supplements

Cosmetics and Fragrances

• Fragrances: Used in perfumes and other cosmetic products.
• Dyes and pigments: Used for coloration.
• Antioxidants and skin-protecting agents: Added to cosmetic formulations.

Agriculture
• Biopesticides: Natural insecticides and fungicides for crop protection.
• Herbicides: Naturally derived weed control agents.

Other Industries:
• Leather Industry: As tanning agents.
 Industrial Applications of Secondary Metabolites

Pharmaceuticals – anticancer, Vincristine & Vinblastine (Catharanthus roseus) –

Alkaloids
antihypertensive, analgesics anticancer; Morphine (Papaver somniferum) – analgesic
Taxol (Taxus brevifolia) – anticancer; Artemisinin
Terpenoids Anticancer drugs, flavors,
(Artemisia annua) – antimalarial; Menthol (peppermint) –
(Isoprenoids) fragrances, insecticides
flavoring
Antioxidants, food preservatives, Anthocyanins (grape skins, berries) – natural food colorants;
Phenolics & Flavonoids
nutraceuticals, colorants Resveratrol – cardioprotective agent
Cardiac therapy, detergents, Digoxin (Digitalis purpurea) – cardiac drug; Saponins – used
Glycosides
insecticides in soaps/detergents
Tannins Leather tanning, dyes, astringents Tannic acid – leather industry, ink production
Lavender oil – perfumes; Clove oil – antiseptic; Eucalyptus
Essential Oils Cosmetics, aromatherapy, flavors
oil – decongestant
Pharmaceutical precursors for
Diosgenin (from Dioscorea spp.) – precursor for steroidal
Steroids contraceptives, anti-inflammatory
drugs
drugs
Polyketides & Pharmaceutical antibiotics, Erythromycin (Saccharopolyspora erythraea); Tetracycline
Antibiotics antifungals (Streptomyces spp.); Amphotericin B – antifungal
Carotenoids (β-carotene, lycopene) – food coloring & vitamin
Pigments Food and textile industry, cosmetics A source; Curcumin (Curcuma longa) – natural dye,
nutraceutical
Adhesives, industrial products, Natural rubber (Hevea brasiliensis); Podophyllotoxin –
Resins & Latex
pharmaceuticals anticancer drug precursor