SECONDARY METABOLITE Play a minor role in basic life processes but have an ecological role.

For example : attractant of pollinators chemical defence against microbes, insects and higher predators. Industrial products, agrichemicals, pharmaceutical and food additives. High value chemicals Dependence on plant continue Limited taxonomic distribution Synthesis under certain conditions Example of cell specialization

Protection against feeding : Coffea

Detoxification of substances accumulated in primary metabolism Coordination of activities of different individuals of same species.

Carotenoid protects chloroplast againt photochemical degradation by intensive radiation.

Pharmaceuticals : alkaloids, steroids , cardinolids

Food and flavours : sweetners, bittering agents , pigments

Plants prefered for following reasons:Structural complexity Novel active compounds detected Model for synthesis of more potent new analouges
Staring compounds for further modifications Cell culture preferred than plants

Round the year production Failure of crops Mass extinction

Compounds produced exclusively in tissue culture and not in intact plant compound Epchrosine Dehydrodiconiferylalcohol--Diglucoside Peniculid A pericine Plant sp. Ochrosia ellipitca Plagiorhegma dubium

Andrographis paniculata Picralima nitida

Examples where cell cultures have produced natural compounds in amounts equal or higher than whole plants Product Yield(% dry weight) Yield(% dry weight) Whole plant Cell culture

ajmalicine anthraquinones berberine caffeine diosgenin

0.3 2.2 2.4 1.6 2.4

1.0 18 13.4 1.6 7.8

Influence of Culture conditions on Secondary metabolite Accumulation Physiological age of Culture Production Conditions Culture conditions a) Culture mediumGrowth and production of sec. metabolites inversely related late stationary phase growth inhibition is associated with cytodifferentiation and induction of enzymes for secondary metabolism Dual culture system 1) Growth medium 2) Production medium Growth medium modification a) reduction or elimination of 2,4-D (auxin) b) reduction of phosphate level c) increase in sucrose level ( C/N) ratio

Decrease in phosphate concentration increases cinnamoil putrescine by nicotinia tobaccum (3x) Increase in copper concentration increases shikonin concentration in lithospermum erythrorhizon Golden apple increased CO2 necessary for apple fragrance. Increased oxygen supply decreases ajmalicine production

pH of medium

- permeability of cell membrane is enhanced which help in release of intracellular alkaloids

Light imp of light for optimal expression of some pathway in cultured

cells (flavanoids)

Selection of high yielding lines- explants

heterogenous w.r.t. sec. metabolite productivity selection of cell linesperiodic selection to maintain high productivity stability- over several cycles

ELICITATION
Reaction characterisitic of its defensive response. Secondary metabolite production increases under stress. ELICITOR : biotic and abiotic Biotic includes : fungal extract (conidia of Verticilium dahlilae improves gossypol production 10 times , 25 fold increase in sanguinarine concentration by Pappver sp. after addition of Botrytis homogenate) Abiotic includes : inorganic and organic chemicals, UV irradiation (NaCl , KCl commonly used elicitors catherantine production increased in Catheranthus roseaus , production of dimeric alkaoid by Catheranthus roseaus increases with irradiation to near UV light)

ORGAN CULTURE
Limitation of undifferentiated plant cell for SMP Genetic instability Differentiation necessary for SMP Organ culture better option (hairy roots, somatic embryo,) example- callii of Digitalis produced cardiac glycoside when induced to differentiate somatic embryos Attropa belladona calli do not roduce tropane hyoscyamin but started producing it with dirrefentiatin of roots Ri plasmid of Agrobacterium rhizogenesis induces hairy root disease -These hairy roots show high degree lateral branches profusion of root growth and absence of geotrophism. -High growth rate -Hairy roots of B. vulgaris produces twice as much 2-3 times more betacyanin and beta xanthin as seedling roots Initial inoculum 2-4 mg (1-2 root tips) showed 2500-5000 fold increase in three weeks 17 days old culture of hairy root of B. vulgaris produced twice as much beta cyanin and three times as much beta xantine as seedling roots. Specialised bioreacter impeller modification

Extraction of S.M.
Intracelluar, rarely extra cellular Destructive and non destructive Destructive Kills cells , cannot be reused Non destructive cells can be used for large no of cycles, reduces cost of production, reversible Permiabilization DMSO application, low pH medium, Sonication with continuous ultra sound electric treatment C. roseus 400-720V/Cm DC ultra sound (1.02 MH2) Betain secretion cells retain capacity to produce pigment 100% alkaloid released by immobilized cells of C.roseus by periodic addition of acid/base

Removal of secreted Products need to be removed

Released products may be degraded Lead to feed back inhibition Materials used
Amberlite XAD-7 for ajmalicine and serpetine AC Reverse Phase Silica are other solid phases Dimethyl siloxane polymer silica based antifoam fluidS

Economic out look

Cell culture production should be economical C. roseus- ajmalicine 3600 kg/year 40 time increase in specific rate of biosynthesis Production of compounds rare , from endangered plants Increase in s.r.s. by high yielding lines , culture condition manipulation, low cost medium components, judicious selection of bioreactors, immobilized cells

Difference between microbial and plant cell characterisitics size Doubling time Growth pattern Fermentation time Oxygen requirements Shear sensitivity Water content Regulatory mechanism Genetic makeup Product accumulation Microbial cell 2-10 m 1 hour Single cells pellets mycelia 2-10 days 10-100 m mol/g/hr insensitive Approx 80% complex stable Often extracellular Plant cell 50-100m 2-6 days clumps 2-3 weeks 1-3 m mol/g/hr sensitive Approx 90% Highly complex May be highly variable Mostly intracellular

LIMITATIONS
Unique structure challenge for aseptic engineering. Interlocked mass Stagnation of liquid flow and coalasces of bubbles- limit mass transfer

REQUIREMENTS OF BIOREACTER (PLANT)

Capacity of oxygen supply and bubble dispersion. ability to reduce hydrodynamic stresses generated inside the reactor. Mixing of culture broth at high cell concentration Ability to control temperature, pH, nutrient concentration. Ability to control aggregate size Easy to scale up Simple to operate, ability to carry out aseptic operation for long duration.

Stirred tank air dispersed by mechanical agitation Shearing stresses generated can be lethal

Modifications
Impeller speed reduced Replacement of impeller by marine screen or paddel Enlargement of sample ports Used for first industrial plant tissue product shikonin Disadvantages High energy requirement and complex const. difficulty in scaling up

Bubble column reactor

Simple gas liquid reactor cylindrical vessel aerated at the bottom gas dispersed through a deep

pool of liquid by means of nozzles or perforated plates

Advantagesfacilitates sterile operation because of absence of moving parts High mass and heat transfer areas without the input of mechanical energy Scale up relatively easy Limitations

Undefined fluid flow pattern resulting in non uniform mixing

Air lift Reactorair is used for aeration and mixing of contents. Based on drought tune principle . Air sparged into base of reactor lowers the density of medium, it rise up and draft tube pulls fresh medium at the base reactor Most suitable for large scale cultivation of plant cells Reasonable mixing, low shear, low contamination Operation cost low than stirred tank Demerits Formation of dead zones due to insufficient fixing especially at high cell densities

Rotating drum reactor Better growth and less hydrodynamic stress

C & D Air lift Reactor, E- Rotating Drum Reactor

IMMOBILIZATION OF CELLS
A production cost due to slow growth of plant cells, low product yield, genetic instability of selected line, low shear resistance of cells and intracellular accumulation of products. IMMOBILIZED CELL CULTURE Enable prolonged use of biomass. Cell density in a bioreactor can be increased 2-4 times enables use of small reactors.

Protection of cells against shear forces.

Separates cells from medium product is extracellular. Uncoupling of growth and product formation

Non dividing immm. cells less prone to genetic changes

Minimizes fluid viscosity which is responsible for improper mixing and aeration Promote secondary metabolite secretion

TYPES OF IMMOBILIZATION

1. Direct intercellular binding- natural affinity 2. Covalent coupling on inert matrices

3. Intercellular connection via bi or poly functional reagent (cross linking)

4. Mixing with suitable materials, changing consistency with temperature (embedding) 5. Physical retention with frmework of diverse pore size and permeability (entrapment, microencapsulation)

choice of immobilization system

Cheap, easy to handle, allow large amount of biomass to be fixed.

IMMOBILIZED PLANT CELL SYSTEM

Limitations two phases should be decoupled initial biomass must be produced in suspension culture secretion of product in external medium is needed. stability of excreted products gel matrix introduces an additional diffusion barriers.

Advantages of immobilized cells

Enable prolonged use of biomass Increases cell density upto 2 4 times Protected against sheared forces Simplify downstream process

BIOTRANFORMATION

Chemical conversion of an exogeneously supplied substance by living cell cultures permeablised cells or entraped enzymes derived from cell culture Single step or multistep Substrate natural or synthetic- novel or already known Versatility of PCC to effect biotransformation (regio and stereo specific) is considerable

Types of reactions epoxidation, esterification, glycolsilation , hydrolysis, isomerization, methylation, Demethylation dehydrogenation.
Examples

Arbutin- skin depigmentation synthesized from Hydroquinone (Cathranthus rosaeus ) less concentration of pre. Addtion of inexpensive precursor into culture at the beginning (6mM) and there after continuously (1.4mM/h)

Podophyllotoxin, a precursor of the semisynthetic anticancer drug, is generally extracted from its source plants Podophyllum hexandrum and P. peltatum [ A cell line of Po. peltatum, active in biosynthesis of podophyllotoxin, was able to maintain repeated biotransformation of butanolide to the podophyllotoxin analogue (50% yield) by oxidative coupling in a bioreactor for more than 15 cycles of 24 when supplied with dibenzylbutanolides Vinblastin and vincrestine need vindoline and cathrenthine.Vindoline (precursor not available or little ) Misawa 1988 produced 3-4 anhydro vinblastin by coupling monomeric alkaloid catheranthine produced by cell and vindoline added externally by cell free enzyme extract of Catheranthus roseaus

Successful Cases
Shikonin and Ginseng Shikonin Antibacterial, anti inflamatory Traditionally from reddish purple roots of L. erythrorhizon 3-4 yrs to root to mature (Shikonin content of root low(1-2%)) Extinction Mitsuicehochemical co. japan Cell culture High yield lines- culture condition Two stage 15% of biomass 200ltr-750 ltrs

Ginseing
Panax ginseing (herb oriental medicine) 4-7 yrs to mature Furuya et al. P. ginseing callus culture Callus 21.1 %, Crowngall 19.3%, Redifferentiated root 27.4% Natural root 4.1% Nitto Denka scale up 1958 government approval

Additive to wine , tonic, drinks, soup.

Culture of Single Cells -

Single cells can be cultured using the following techniques:

(1) Filter paper raft- nurse tissue technique

(2) Microchamber technique (4) Bergman's plating technique

filter paper raft- nurse tissue technique

microchamber technique

Bergman's plating technique