Transcript No Slide Title

Lecture 36&37
PLANTS AS BIOREACTORS
WHAT IS A BIOREACTOR ?
• A device in which a substrate of low value is
utilised by living cells to generate products of
higher value.
• Plants are exploited as bioreactors for the
production of biomolecules.
WHY PLANTS ARE USED AS BIOREACTORS ?
•
Post translational modifications
•
Storage costs
•
Ethical considerations
COMPARISON WITH OTHER PRODUCTION SYSTEM
• Low cost alternative
• Post translational modifications
• Storage facilities
• Low upstream production cost
PRODUCTION OF BIOMOLECULES
Carbohydrates
1) Cyclodextrin biosynthesis
• Starch acts as the substrate
• Bacterial gene encoding cyclodextrin
glycosyl transferase (cgt )
Contd...
Cyclodextrin glycosyl transferase
Contd...
HOW GENE CONSTRUCT IS MADE FOR cgt?
(Cyclodextrin glycosyl transferase)
a) Patatin gene promoter (tuber specific)
b) Sequence encoding peptide of RUBP carboxylase
c) cgt gene from Klebsiella pneumoniae
d) 3' sequence of nos gene of Agrobacterium
Contd...
Gene construct of cgt gene
5'
3'
P
c
p
t
P
Patatin promoter
c
cgt gene
p
Sequence encoding transit peptide of
RuBP carboxylase
t
nos terminator
Contd...
• Targeted to amyloplast
• Expressed in tubers
• Expression level : 0.0010.01%
(Goddijn and Janpen, 1995)
Amyloplast
2) Increasing Starch accumulation
ADP- Glucose pyrophosphorylase
ADP- Glucose
Starch
•
A mutated bacterial gene (glgc16) encoding
ADP glucose pyrophosphorrylase
•
Expressed in potato tubers
•
Targeted to amyloplasts
•
60% increase in starch than control
( Verisser and Jaciobsen, 1993 )
Rerouting the starch to produce fructan
•
Fructosyl transferase gene from Bacillus subtilis
•
Introduced in tobacco and potato plants
•
Accumulation level:
3-8% of dry wt. In tobacco leaves
1-30% in potato leaves
1-7% in potato microtubers
Metabolic Engineering of Carbohydrate Metabolism
(Goddijn and Janpen,1995)
PRODUCTION OF PROTEINS FROM PLANTS
Expression of peptide relies on:
a) Stable intergration of transgene
b) By transient expression of genetically
engineered viruses
HUMAN THERAPEUTIC PROTEIN-SOMATOTROPIN (hST)
• Synthesis of hST and ubiquitin fusion genes
• Cloning of chimeric hST genes using pPRV
vectors
• Introduction to tobacco leaf chloroplast by
biolistic process
• Leaves with different ages show different
hST accumulation
( Jeffrey et al., 2000 )
SEEDS AS BIOREACTORS
Seeds
Why seeds are used as bioreactors ?
• Storage facility
• Transportation
• Oral consumption
• Existing agricultural
facilities in seed handling
(Sun et al., 2002)
Production of recombinant Hirudin
from seeds
• Hirudin has antithrombin
activity
• Isolated from Hirudo
medicinalis
• Limited availability (1 leech
head contains
20mg of
hirudin)
3- D View of Hirudin
• Construction of synthetic
gene with amino acid
sequence of hv2
Contd...
Oleosin - Hirudin fusion gene construct
5'
3'
P
o
X
H
o
Oleosin gene
x
Cleavage site
H
Hirudin gene
t
nos terminator
t
Contd...
• Fusion of gene with
Arabidiopsis oleosin gene
• Introduction of fusion gene
construct into Brassica napus
• Expression of seed specific
oleosin hirudin transcripts
• Protein purified by oleosin
partition technology
( Dana et al., 1996 )
Brassica napus
Contd...
Oleosin based purification of heterologous polypeptides
(Goddijn and Janpen, 1995)
PRODUCTION OF BRYODIN IN
TOBACCO PLANT
• Tobacco plants that are able to produce
bryodin.
• This protein, which is produced in the roots of
bryonia, deactivates ribosomes and is being
tested for its effect against HIV infection.
Plant Transformation
1.
2.
The plant leaf disc is dipped in a
solution of bacteria. The bacterial
"Trojan Horse" infects the edges of
the leaf disc and in the process
integrates the pharmaceutical
protein gone into the plant genome
(pict 1).
After infection the discs are placed
on selection media that a flows only
plant cells that carry the protein
gene to survive and regenerate into
plantlets. After about six weeks on
selection media, a large number of
plantlets that carry the
pharmaceutical protein gene are
visible at the edges of the original
leaf disc (pict 2 & 3).
3. The plantlets are
removed from the leaf
disc and placed in clear
plastic boxes that contain
media that allows them
to form roots (pict 4).
4. The rooted plantlets are
placed in pots and plants
are allowed to grow and
produce seed. This seed
can then be used for
large scale production of
the pharmaceutical
protein (pict 5).
Protein Trafficking
Following translation of
the molecular ring
gene, the protein will
move through the
endoplasmic reticulum
and Golgi apparatus
for processing, folding
and glycosylation.
PRODUCTION OF SPIDER
SILK PROTEINS IN PLANTS
Production of transgenic plants
Spider silk protein
Expression of spidroin-ELP-fusion proteins
in the ER of transgenic plants
Purification of spider silk-ELP
fusion proteins
From transgenic plants
Spidroin-ELP-fusions
could be purified by
addition of salt and by
heat to 95% purity.
BIOPHARMACEUTICALS FROM PLANTS
• Plants constructed to express proteins like
α-interferon, human serum albumin etc.
• Two expensive drugs are produced from plants
A) Glucocerebrosidase
B) Granulocyte macrophage colony stimulating
factor
GLUCOCEREBROSIDASE
• Lysosomal hydrolase
• Cause Gaucher’s disease
• Earlier this enzyme was purified from human placentas
• Now synthesized from tobacco plants
(Giddings et al., 2000)
Production of human lysosomal
enzymes in Nicotiana tabacum
• Gluco cerebrosidasegaucher disease
• Alphahexosaminidase- TaySachs disease
• Alpha-L-iduronidaseHurler syndrome
INDUSTRIAL ENZYMES
Cellulase
• Isolated from bacterial
and fungal organisms
• Expressed in potato
plants
• Enzymes produced in
foliage and vines
Phytase
• Isolated from Aspergillus niger
• Expressed in seeds
• Replace feed supplements for broiler chicken
PRODUCTION OF BIOMOLECULES FROM PLANTS
S No
Compound
Origin of gene(s)
Appl.
Plant sp.
Oilseed rape
Arabidopsis, oil
seed rape,
soybean
LIPIDS
Mediumchain fatty
acids
Monounsaturated
fatty acids
California
baytree
Rat
Food,
detergent,
industrial
Food
3.
Polyhydroxy
butyric acid
Alcaligenes
eutrophus
Biodegradable
plastics
4.
Saturated
fatty acids
Brassica rapa
Food,
Oilseed rape
confectionaries
1.
2.
Tobacco
CARBOHYDRATES
5. Amylose free
starch
Solanum
tuberosum
Food,
industrial
Potato
6. Cyclodextrins
Klebsiella
pneumoniae
Food,
Potato
pharmaceutical
7. Fructans
Bacillus
subtilis
Industrial, food Tobacco,
potato
8. Increased amount
of starch
Escherichia
coli
Food,
industrial
9. Trehalose
E.coli
Food stabilizer Tobacco
Potato
10.
PHARMACEUTICAL
POLYPETIDES
Antibodies
11.
Mouse
Various
Mainly
tobacco
Antigens
Bacteria,
viruses
Orally
administered
vaccines
Tobacco,
tomato,
potato,
lettuce
12.
Growth hormone
Trout
Growth
stimulation
Tobacco,
Arabidopsis
13.
Hirudin
Synthetic
Thrombin
inhibitor
Oil seed
rape
14.
Human serum albumin
Human
Plasma
expander
Tobacco,
potato
15.
Interferon
Human
Anti-viral
Turnip
INDUSTRIAL
ENZYMES
Alpha-amylase
Bacillus
licheniforms
Liquefaction
of starch
Tobacco,
alfalfa
Phytase
Aspergillus
niger
Animal feed
Tobacco
Xylanase
Animal feed,
Clostridium
thermocellum, paper and
pulp, baking
Cryptcoccus
albidus
Tobacco
PLANT CELL SUSPENSION CULTURE AS
BIOREACTORS
•
Secondary metabolites & recombinant
proteins - production
•
Antitumour agents like taxol can be
produced
•
Taxol from Taxus sp - treatment of breast and
ovarian cancers
(Seki et al., 1997)
ADVANTAGES OF PLANTS AS BIOREACTORS
• Can produce high level of safe homogenous
functional biomolecules
• Modern agriculture practice - easy scale up and
processing
•
Easy storage
ADVANTAGES OF PLANTS AS BIOREACTORS
• Chimeric plant virus can be used to produce
vaccines
•
Administration safe and painless
•
Long shelf life (seeds)
•
Low cost
LIMITATIONS AND REMEDIES
Limitations
* Downstream
processing expensive
and difficult
* Accumulation of
transgene product in
plant
Remedies
* Expressing the protein
with sturctured oil body
protein
e.g: Hirudin
* Targeting the transgene
into proper cellular
compartment
FUTURE CHALLENGES
• Engineering challenges like maximization of expression
levels
• Environmental safety
• Stability of product under storage
• Evaluation of dosage requirement
• Regulatory considerations and legal standards