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?
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
(Cyclodextrin glycosyl transferase)
Contd...
5' 3'
Patatin promoter
cgt gene
Sequence encoding transit peptide of
RuBP carboxylase
nos terminator
P c p t
Gene construct of cgt gene
P
c
p
t
Contd...
• Targeted to amyloplast
• Expressed in tubers
• Expression level : 0.001-0.01%
(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 )
• 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
Rerouting the starch to produce fructan
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
• Storage facility
• Transportation
• Oral consumption
• Existing agricultural facilities in seed handling
(Sun et al., 2002)
Why seeds are used as bioreactors ?
See
ds
Production of recombinant Hirudin from seeds
• Hirudin has antithrombin activity
• Isolated from Hirudo medicinalis
• Limited availability (1 leech head contains 20mg of hirudin)
• Construction of synthetic gene with amino acid sequence of hv2
Contd...
3- D View of Hirudin
P o X H t
o
x
H
t
Oleosin gene
Cleavage site
Hirudin gene
nos terminator
Oleosin - Hirudin fusion gene construct
Contd...
5' 3'
• 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 Transformation1. 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).
2. 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 TraffickingFollowing 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 cerebrosidase- gaucher disease
• Alpha-hexosaminidase- Tay-Sachs disease
• Alpha-L-iduronidase- Hurler 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
S No Compound Origin of gene(s) Appl. Plant sp.
1.
LIPIDS
Medium-chain fattyacids
Californiabaytree
Food,detergent,industrial
Oilseed rape
2. Mono-unsaturatedfatty acids
Rat Food Tobacco
3. Polyhydroxybutyric acid
Alcaligeneseutrophus
Biodegradableplastics
Arabidopsis, oilseed rape,soybean
4. Saturatedfatty acids
Brassica rapa Food,confectionaries
Oilseed rape
PRODUCTION OF BIOMOLECULES FROM PLANTS
5.
CARBOHYDRATES Amylose free starch
Solanum tuberosum
Food, industrial
Potato
6. Cyclodextrins Klebsiella pneumoniae
Food, pharmaceutical
Potato
7. Fructans Bacillus subtilis
Industrial, food Tobacco, potato
8. Increased amount of starch
Escherichia coli
Food, industrial
Potato
9. Trehalose E.coli Food stabilizer Tobacco
10.
PHARMACEUTICALPOLYPETIDESAntibodies Mouse Various Mainly
tobacco
11. Antigens Bacteria,viruses
Orallyadministeredvaccines
Tobacco,tomato,potato,lettuce
12. Growth hormone Trout Growthstimulation
Tobacco,Arabidopsis
13. Hirudin Synthetic Thrombininhibitor
Oil seedrape
14. Human serum albumin Human Plasmaexpander
Tobacco,potato
15. Interferon Human Anti-viral Turnip
INDUSTRIALENZYMES
Alpha-amylase Bacilluslicheniforms
Liquefactionof starch
Tobacco,alfalfa
Phytase Aspergillusniger
Animal feed Tobacco
Xylanase Clostridiumthermocellum,Cryptcoccusalbidus
Animal feed,paper andpulp, baking
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 Remedies
* Downstreamprocessing expensiveand difficult
* Expressing the proteinwith sturctured oil bodyprotein e.g: Hirudin
* Accumulation oftransgene product inplant
* Targeting the transgeneinto proper cellularcompartment
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
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