plant as bioreactor
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PLANT AS BIOREACTOR
Submitted by : Arun & Udhaya
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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 : Biomolecules, industrial, chemical & pharmaceutical
etc.
Number of products are successfully produced in plants using this ‘molecular
farming’
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Plant as Bioreactor
Comparison with other production system
Fast biomass build up
Post translational modifications
Easy storage and distribution
Low upstream production cost
Transgenic animals as production systems: Public and ethical concerns
Types of Plant bioreactors
Seed-based plant bioreactors
Plant Suspension Cultures
Hairy Root System Bioreactor
Chloroplast bioreactor
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Plant as Bioreactor
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.
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Plant as Bioreactor
Key process…
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
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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.Streatfield SJ et al., 2003
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Plant as Bioreactor
Year Recombinant protein Source Importance Reference
1989 Mouse immunoglobulin
Tobacco1st 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
2006HN proteins of Newcastle disease virus
Rice 1st plant-based vaccine (for poultry) approved by the USDA
Dow AgroSciences, 2006
2007 Antibody against Hepatitis B
Tobacco1st commercialized plant-derived antibody (marketed in Cuba)
Pujol et al., 2007
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…
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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)
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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?
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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
Tree depicting biotechnological advances using plants as bioreactorsSource: www.plantbioreactor.co.in/images/00_112.jpg
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Plant as Bioreactor
Products : 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.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.
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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.
Schillberg et al., 1999, Ma et al., 2003, Goldstein and Thomas, 2004
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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)
Sub cellular destination - high level expression.
Further research is concentrated on Humanizing the Plant N-glycans.
Schillberg et al., 1999, Ma et al., 2003, Goldstein and Thomas, 2004
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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).
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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)
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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.
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Plant as Bioreactor
Conclusion (Cont.…)
Table source: Yansong miao et al., 2008
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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.
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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.
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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.
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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.