Molecular Farming in Plants

Dr. Neelu Joshi

Assistant Professor
Department of Biotechnology and Bioinformatics
Padmashree Dr. D.Y. Patil University
Navi Mumbai

Applications of r DNA
technology in Agriculture

Crop protection against various biotic and abiotic

stresses
Modification of production traits /introducing novel
traits in crops
Improving colour and smell of flower, taste of food
crops
Modifying starch, protein and oil content
Facilitating the accumulation of vitamins and
minerals in food
Altering the properties of fruits and vegetable
Introducing male sterility
Increasing the levels of total soluble solids
Metabolic engineering
Molecular farming

Molecular Farming
Application of genetic engineering techniques to

the synthesis of commercial products in plants.

It includes:

-enhanced production of products that are

already
extracted from plants.
-manufacture of compounds novel to plants.

Molecular farming in
plants
Potential vehicles for protein production:
for bulk production- leaves of tobacco &alfalfa,
for edible products- tomato, potato, maize,
wheat, rice
Production strategies
Stable expression: constitutive and tissue
specific
Transient expression: using plant virus (Cp
gene)as vector

Molecular farming for carbohydrate

Polyfructans (G-F-Fn)
Soluble polymer of fructose, synthesized and stored in vacuole
Fructans are used as carbohydrate reserves
Ex. Inulins-major storage carbohydrate found in onion bulbs
Levans-leaves and stem of grasses
Glucose-fructose-(fructose)n i.e. G-F-Fn
Biosynthetic pathway of fructans
1. G-F+G-F = G-F-F+G
SST enzyme
2. G-F-Fn+G-F-F = G-F-Fn+1 +G-F FFT enzyme.
Sucrose is the initial acceptor molecule of the chain, so the first
sugar in the fructan chain is always glucose. However, for
each remaining fructose residue added to the chain a glucose
residue is released, which in plants is transported back out of
the vacuole into the cytosol.
sacB gene(produces levansucrase in one step) of B. subtilis
+vacuolar targeting sequence from yeast, in tobacco.
Sac B (B.amyloliquifaciencs) +zein promoter +vacuolar
targetting seq. expressed in maize endosperm

Fructanbeet- transgenic
sugarbeet
Production of short oligofructans GF2, GF3, GF4.
Gene for enzyme 1-SST from Jerusalem artichoke
Applications:
1.In neutraceutical market
2.As substitute of sugar in foods and drinks
3.Low-calorie sweetner
4.Encourage growth of beneficial gut flora and

cause acidification of the digestive tract, which

inhibits E.coli.

Molecular farming for lipids Polyhydroxyalkanoates (PHAs) form bioplastics

Bioplastics
Currently produced by microbial fermentation
Polyhydroxybutyrate (PHB)-best characterized PHA found as

intracellular inclusions in bacteria e.g. Alcaligenes eutrophus.

Biosynthetic pathway in A.c.
Pha B
Pha
Acetyl-CoA Pha
acetoacetyl coA
3-hydroxybutyrl-coA
c
A
PHB
Cytoplasm
plastid
Pha
Acetyl-CoA
Acetyl-CoA
A
Acetoacetyl coA Pha
Lipids
acetoacetyl
B
Pha B
coA
Pha
Isoprenoids 3-hydroxybutyrl-coA
3Pha c
C
hydroxybutyrl-coA
PHB
A.thaliana
PHB
CaMV+TPP+N Rubisco+Gene+T
Approaches: gene pyramiding, triple construct

Molecular farming for

proteins
Antibodies
Subunit vaccines

Plantibodies:
in
plants
1.
First functional Ab:

Antibodies produced

mouse IgG1 (1989)

T1 (-heavy chain) X T2( -light chain)

T3 (F1)
Nascent chains targetted to ER lumen for processing and assembly of Ab
2.ScFv:most commonly used and most successful
variable regions of H and L chains are responsible for antigen binding
Seq. L chain variable region + Seq. H chain variable region

synthetic gene
3.Secretory Ab (sIgA):protection against dental caries caused by S.
mutans
:Microbial infections at mucosal
sites
T1 (H)X T2 (L)X T3 (J) XT4 (Sc)
Topical application on human teeth & prevented colonization of S.
mutans upto 4 months.
Effective as produced in murine hybridoma,effective binding to antigen.

Recombinant Hirudin
Anticoagulant peptide (~7-kDa)in salivary glands

of Hirudo medicinalis.
Most potent inhibitor of thrombin-high
therapeutic value in the treatment of thrombosis.
Advantages over heparin
-Low immunogenecity
-doesnt require endogenous cofactor for
activity
- does not react with other proteins in the
blood.

Recombinant Hirudin in Brassica napus

P-O Oleosin gene Hirudin gene

Endoprotease factor Xa
cleavage site
Expression in transgenic plant protein targeted to oil bodies
Crush seeds
Spin
Oil body
Remove ,spin, treat with protease

protein in aq. phase

Recombinant Vaccines
Edible vaccines: It can be produced for those

antigens which illicit an immune response

when ingested orally.