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PLANT AS
BIOREACTOR
Submitted by : Komal
Verma
Roll no.3599
1
Plant as Bioreactor
Introduction
A device in which a substrate of low value is utilized by living cells to
generate products of higher value.
From earlier days - Microbes & animal cell culture used to produce
biomolecules.
Advancement in Plant genetic engineering : Possible to use as Bioreactors.
Plants are important food resources from the earliest times.
Plants as bioreactors :Plants are genetically engineered to make products
that are not of plant origin (Therapeutically & industrially important
products, Biomolecules, chemical & pharmaceutical etc.
2
Plant as Bioreactor
Why plants are used as bioreactors?
Fast biomass build up
Post translational modifications
Easy storage and distribution
Low upstream production cost
Lacking of contamination by animal pathogen.
Advantages: Plant as bioreactor
Low cost source.
Simple & Cost effective.
Plant pathogens do not infect humans or animals.
Easy scale up & rapid harvesting.
Chimeric plant viruses can be used in production of vaccines.
Produce large biomass.
Easy storage for long time
3
Plant as Bioreactor
Key process…
4
Design gene for high level
expression
Plant transformation
Regeneration of Cell
Selection of transgenic
Growth of plants in field
Harvesting of plant materials
Purification of product
Biosafety & Functionality
test
Plant as Bioreactor
Types of PLANT BIOREACTORS
5
plant
bioreactors
Seed based
plant
bioreactors
Plant
suspension
culture
Hairy root
system
bioreactor
Chloroplast
bioreactor
Protein
storage
vacuoles
Oil bodies
Seed based plant bioreactors
• Seeds provide a suitable
environment
• High rate of protein synthesis
• Accumulate large amount of
proteins during seed development.
• Eg. ά-L-iduronidase in
Arabidopsis thaliana seeds.
Seed based plant bioreactors
• expression is controlled using seed specific
promoters
• For eg. in maize globulin-1, and in rice
glutelin promoter Gt-1.
• Two types
• Seed Protein Storage Vacuole Bioreactors
• Seed Oil Body Bioreactors
• Main advantage : proteins do not degrade.
Seed Protein Storage Vacuole
Bioreactors
• Protein storage vacuoles (PSVs)
• compartments for the storage of recombinant
proteins in seed bioreactors
• three distinct sub compartments:
• the matrix, globoid and crystalloid.
• matrix :soluble storage proteins
• globoid : hydrolytic enzymes.
• crystalloid : BP-80 TMD and the CT of alpha-
tonoplast intrinsic protein sequences.
Transporting pathways of
subcompartments
• For eg : transmembrane domain (TMD) + cytoplasmic tail
(CT) of BP-80 a reporter from prevacuolar
compartment (PVC) to PSV globoid
• BP-80 TMD + CT of alpha-tonoplast intrinsic protein (TIP)
sequences target the same reporter to PSV crystalloid in
transgenic tobacco seeds
• Similarly, AFVY, a vacuolar sorting determinant of
phaseolin, can deliver reporter or recombinant protein to
PSV matrix.
Seed Oil Body Bioreactors
• seed oil body (OB) are surrounded by oleosin proteins that function in maintaining the structural integrity of OBs.
• provides a recognition signal for lipase binding during oil mobilization in seedlings.
• can store large amount of macromolecules
• Eg. manufacture of the anticoagulant hirudin in the oil body of seeds Brassica napus and Brassica carinata
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 )
•an anti-coagulant for treatment of
thrombosis in rapeseed by SemBioSys(Boothe et al., 1997)
Brassica napus
Contd...
Oleosin based purification of heterologous polypeptides
(Goddijn and Janpen, 1995)
Plant as Bioreactor
Oil bodies
PLANT SUSPENSION CULTURES
• Provide a fast system for producing secondary
metabolites, biologically active recombinant proteins
and antibodies.
• The recombinant proteins sub cellular organelles or
into extra cellular space
• Eg: expression of 80-kDa human lysosomal protein
(controlled by 35s CaMV and a signal peptide) in
transgenic tobacco BY-2 cells culture media.
• Antitumour agents like taxol can be produced from
Taxus sp - treatment of breast and ovarian cancers.
Hairy Root System Bioreactor
• The Hairy Root System with its rhizosecretion
is due to infection of the soil bacterium-
Agrobaterium rhizogenes.
• biosynthetic stability
• suitable for making biopharmaceuticals
• Eg : scopolamine in Hyoscyamus muticus L.
hairy root culture
Chloroplast bioreactor
• Insulin, interferons and other biopharmaceutical proteins can be made using Chloroplast bioreactor.
• One method : foreign genes are inserted into nuclear chromosomes and then target the expressed proteins into chloroplast.
• Other: direct targeting in chloroplast genome
• Eg: high yield in the expression of human serum albumin protein in chloroplast.
Plant as Bioreactor
Production platforms
Cereals: Maize, Rice, Barley and Wheat are used.
o High seed protein content (7 to 10%).
o High biomass yield.
o Ease of transformation.
o Ease of scale-up.
Major disadvantage of Maize is: Cross pollination
But rice has advantage of self-pollination.
Rice as bioreactors : Oral delivery system for vaccine antigens,
immunotherapy and therapeutic proteins are recent advances.
19
Streatfield SJ et al., 2003
Plant as Bioreactor
Year Recombinant protein Source Importance Reference
1989 Mouse
immunoglobulinTobacco
1st report on the production of antibody in
plants.Hiatt et al., 1989
1997 Chicken avidin Maize 1st commercialized plant-derived protein. Hood et al., 1997
2003 Bovine Trypsin Maize 1st marketed plant-derived protein. Woodard et al., 2003
2006
HN proteins of
Newcastle disease
virus
Rice 1st plant-based vaccine (for poultry) approved
by the USDA
Dow AgroSciences,
2006
2007 Antibody against
Hepatitis BTobacco
1st commercialized plant-derived antibody
(marketed in Cuba)Pujol et al., 2007
20
Ventria Bioscience (US Based Company) : Two rice-derived proteins
Human lactoferrin and lysozyme
Have received regulatory approvals, and lactoferrin has already been marketed in
bulk for bioprocessing.
Production platform Cont…
Plant as Bioreactor
Production platforms (Cont.…)
Legumes:
Soybean and pea: Seeds have high protein content (20–40%).
Both self-pollinating plants & low risk of contamination.
Soybean : Express a humanized antibody against Herpes simplex virus.
Bovine casein, and a Human growth hormone. (Russell et al., 2005)
Oil seeds:
Safflower and rapeseed : Rich in seed oil & inexpensive downstream.
Safflower : High protein yield, Low acreage, and Self-pollinating.
Oilseed-derived protein : Commercial production of Hirudin, an anti-
coagulant for treatment of thrombosis in rapeseed by SemBioSys (Boothe et
al., 1997)
21
Plant as Bioreactor
a. Shown are various Intracellular
organelles or Extracellular
spaces (ES) that can be used to
store the recombinant proteins
expressed in a plant bioreactor.
b. Targeting strategies in plant
bioreactors.
G - Golgi; PSV - Protein storage vacuole;
OB - Oil body; C - Chloroplast;
ES - Extracellular space;
PVC - Prevacuolar compartment.
Where they are produced?
22
Plant as Bioreactor
Products obtained…
Plants genetically engineered to make products that are not of
plant origin
Products:
Vaccines antigens
Therapeutics products
Nutritional components
Industrial products
Bio plastics
23
Tree depicting biotechnological advances using plants as bioreactors
Source: www.plantbioreactor.co.in/images/00_112.jpg
S No Compound Origin of gene(s) Appl. Plant sp.
1.
LIPIDS Medium-chain fatty acids
California baytree
Food, detergent, industrial
Oilseed rape
2. Mono-unsaturated fatty acids
Rat Food Tobacco
3. Polyhydroxy butyric acid
Alcaligenes eutrophus
Biodegradable plastics
Arabidopsis, oil seed rape, soybean
4. Saturated fatty 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.
PHARMACEUTICAL POLYPETIDES Antibodies
Mouse
Various
Mainly tobacco
11. 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 Clostridium thermocellum, Cryptcoccus albidus
Animal feed, paper and pulp, baking
Tobacco
Products : Plant as bioreactor
PRODUCTION OF BIOMOLECULES
Carbohydrates
1) Cyclodextrin biosynthesis
• Starch acts as the substrate
• Bacterial gene encoding cyclodextrin glycosyl transferase
(cgt ) And Targetted to amyoplast.
•Expressed in tubers.
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...
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
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.
Plant as Bioreactor
Vaccine antigens:
It is an antigenic preparation : Immunity against a disease.
Recent Development : “Edible Vaccines” now more popular as “Plant Vaccines”
From Seeds, freeze-dried fruits and leaves: powder form vaccines are produced.
Antigenic determinants for Pathogens causing diseases have been produced
from plants. (Diarrhea, anthrax, rabies, cancer, HIV, tuberculosis etc)
Antigens like Insulin, rotavirus enterotoxin, anthrax lethal factor, HIV antigen,
foot and mouth disease virus antigen, heat stable toxin have been produced in
plants as a fusion partner of CTB or LTB.
34
http://www.dowagro.com/uk/media/General/20061017.htm, Khandelwal et al., 2003; Sharma et al., 2004, Streatfield and
Howard, 2003, Tiwari et al., 2009 and Youm et al., 2008.
Plant as Bioreactor
Products : Plant as bioreactor
Therapeutic products:
Many form of recombinants antibodies are produced in plants.
• (Full size recombinant antibody, chimeric antibody, secretory antibody)
• Recombination anti-sperm antibody (RASA)
• Gene : scFv1 9 from horse
• Expressed in transgenic tobacco.
• an anti-Salmonella enterica single-chain variable fragment (scFv)antibody in
transgenic tobacco
Sub cellular destination - high level expression.
Further research is concentrated on Humanizing the Plant N-glycans.
35
Schillberg et al., 1999, Ma et al., 2003, Goldstein and Thomas, 2004
Plant as Bioreactor
Products : Plant as bioreactor (Cont.…)
Therapeutic products:
Diagnostic proteins (antibodies and enzymes), replacement
proteins, immune system stimulator/suppressants, biopolymers
and adhesive proteins are produced in plants.
Production of immunoglobulin fragments and their assembly in
plants –reported in tobacco.
Recently known as “plantibodies” – immunochromatography or
medical therapy.
36
Schillberg et al., 1999, Ma et al., 2003, Goldstein and Thomas, 2004
Plant as Bioreactor
Products : Plant as bioreactor (Cont.…)
Nutritional components:
It can provide most of the nutrients required in the human diet.
Plant have been engineered to increase accumulation of:
• β-carotene (Naqvi et al., 2009), Lycopene (Fraser et al., 2002), Flavonoid (Butelli et al., 2008),
Nutraceuticals (Kang et al., 2009), Fatty acid (Hoffmann et al., 2008), Vitamins (Nunes et al., 2009),
Minerals (Lee & An, 2009) & Carbohydrates (Regina et al., 2006).
Biodegradable plastics:
Polyhydroxyalkanoates: biodegradable polymers which occur naturally in plants.
• Plant was engineered to produce PHAs or PHBs in the various plant cell compartments.
When PHB expression targeted to cytoplasm, accumulation level was low.
Expression was increased by targeting plastids, (40% of dry weight was obtained).
37
Plant as Bioreactor
Products : Plant as bioreactor (Cont.…)
Industrial products:
First produced protein : Human growth hormones in tobacco.
Most expensive Drug – hGC (Human glucocerebrosidase) in tobacco (Kaiser
2008).
hST (Human somatotropin) was produced in tobacco (Staub et al. 2000).
Which treats : Turner syndrome, Chronical renal failure & Dwarfism in Children.
rHLF (Recombinant human lactoferrin) : Produced from dehusked rice
grain (Nandi et al., 2002). Which is identical to native HLF.
Synthetic fiber: Produced from Potato and tobacco. (Scheller et al., 2001)
38
LIMITATIONS AND REMEDIES
Limitations Remedies
* Downstream processing expensive and difficult
* Expressing the protein with sturctured oil body protein e.g: Hirudin
* Accumulation of transgene product in plant
* 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.
Plant as Bioreactor
Conclusion…
More and more uses of plant bioreactors are coming up these
days.
Plant bioreactors : Investigated for making enzymes (Suitable for Food).
Another use of plants is to make genetically engineered plants that can
produce seeds which can function as a delivery mechanism for various
industrial enzymes.
As you can see these processes go far beyond the application of
biotechnology in traditional agriculture, and so today,
transgenic plants can produce on a mass scale proteins for
agricultural, veterinary and pharmaceutical use.
41
Plant as Bioreactor
Conclusion (Cont.…)
42
Table source: Yansong miao et al., 2008
Plant as Bioreactor
Problems need to be addressed
Storage issues related to transgenic fruits or leaves.
Most inserted genes are expressed at very low level in plants.
Enhancing the stability of products obtained.
Standardization of dosage in case of edible vaccine.
Examining issues related to commercialization.
Issues relating to the ethical, social, biosafety and environmental
impact.
Some plants produce allergenic compounds.
43
Plant as Bioreactor
References
• Boothe JG, Parmenter DL, Saponja JA. (1997) Molecular farming in plants: oilseeds as vehicles for the production of
pharmaceutical proteins. Drug Dev Res, 42:172–81.
• Butelli E, Titta L, Giorgio M, Mock HP, Matros A, Peterek S, (2008). Enrichment of tomato fruit with health-promoting
anthocyanins by expression of select transcription factors. Nat Biotechnol, 26:1301–8.
• Dow AgroSciences. Dow AgroSciences achieves world's first registration for plant-made vaccines. 2006 Press release
(http://www.dowagro.com/animalhealth/resources/news/20060131b.htm).
• Fraser PD, Romer S, Shipton CA, Mills PB, Kiano JW, Misawa N (2009). Evaluation of transgenic tomato plants
expressing an additional phytoene synthase in a fruit specific manner. Proc Natl Acad Sci USA , 99: 1092–7.
• Goldstein DA, Thomas JA. (2004), Biopharmaceuticals derived from genetically modified plants. QJM, 97: 705–16.
• Hiatt A, Cafferkey R, Bowdish K. (1989), Production of antibodies in transgenic plants. Nature, 342: 76–8.
• Hoffmann M, Wagner M, Abbadi A, Fulda M, Feussner I. (2008), Metabolic engineering of omega3-very long chain
polyunsaturated fatty acid production by an exclusively acyl-CoA-dependent pathway. J Biol Chem, 283: 22352–62.
• Hood EE, Witcher DR, Maddock S, Meyer T, Baszczynski C, Bailey M, (1997), Commercial production of avidin from
transgenic maize: characterization of transformant, production, processing, extraction and purification. Mol Breed, 3:
291–306.
44
Plant as Bioreactor
References(Cont.…)
• Kang K, Lee K, Sohna S, Parka S, Lee S, Kima S, (2009), Ectopic expression of serotonin hydroxycinnamoyltransferase
and differential production of phenylpropanoid amides in transgenic tomato tissues. Sci Hortic, 120: 504–10.
• Khandelwal A, Sita GL, Shaila MS. (2003), Expression of hemagglutinin protein of rinderpest virus in transgenic
tobacco and immunogenicity of plant-derived protein in a mouse model. Virology, 308: 207–15.
• Lee S, An G. (2009), Over-expression of OsIRT1 leads to increased iron and zinc accumulations in rice. Plant Cell
Environ, 32: 408–16.
• Ma JK, Drake PM, Christou P. (2003), the production of recombinant pharmaceutical proteins in plants. Nat Rev Genet,
4: 794–805.
• Naqvi S, Zhu C, Farre G, Ramessar K, Bassie L, Breitenbach J, (2009), Transgenic multivitamin corn through
biofortification of endosperm with three vitamins representing three distinct metabolic pathways. Proc Natl Acad Sci
USA, 106: 7762–7.
• Nunes AC, Kalkmann DC, Aragão FJ.(2009), Folate biofortification of lettuce by expression of a codon optimized
chicken GTP cyclohydrolase I gene. Transgenic Res, doi:10.1007/s11248-009-9256-1.
• Pujol M, Ramírez NI, Ayala M, Gavilondo JV, Valdés R, Rodríguez M, (2005), An integral approach towards a practical
application for a plant-made monoclonal antibody in vaccine purification. Vaccine, 23: 1833–7.
45
Plant as Bioreactor
References (Cont.…)
• Regina A, Bird A, Topping D, Bowden S, Freeman J, Barsby T, (2006). High-amylose wheat generated by RNA
interference improves indices of large-bowel health in rats. Proc. Natl. Acad. Sci. USA, 103: 3546–51.
• Russell DA, Spatola LA, Dian T, Paradkar VM, Dufield DR, Carrol JA, (2005). Host limits to accurate human growth
hormone production in multiple plant systems. Biotechnol Bioeng, 89: 775–82.
• Schillberg S, Zimmermann S, Voss A, Fischer R. (1999), Apoplastic and cytosolic expression of full-size antibodies
and antibody fragments in Nicotiana tabacum. Transgenic Res. 8:255–63.
• Sharma AK, Jani D, Tyagi AK. (2004), Transgenic plants as bioreactors. Ind J Biotechnol. 3: 274–90.
• Streatfield SJ, Howard JA. (2003), Plant-based vaccines. Int J Parasitol, 33: 479–93.
• Tiwari S, Verma PC, Singh PK, Tuli R. (2009), Plants as bioreactors for the production of vaccine antigens. Biotech
adv, 449–67.
• Woodard SL, Mayor JM, Bailey MR, Barker DK, Love RT, Lane JR, (2003), Maize (Zea mays)- derived bovine
trypsin: characterization of the first large-scale, commercial protein product from transgenic plants. iotechnol
Appl Biochem, 38: 123–30.
• Youm JW, Jeon JH, Kim H, Kim YH, Ko K, Joung H. (2008). Transgenic tomatoes expressing human beta-amyloid
for use as a vaccine against Alzheimer's disease. Biotechnol Lett, 30: 1839–45.
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Plant as Bioreactor
THANKS FOR YOUR ATTENTION!
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