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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
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

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.

Plant as Bioreactor

Key process
Design gene for high level expression Plant transformation Regeneration of Cell

Harvesting of plant materials

Growth of plants in field

Selection of transgenic

Purification of product

Biosafety & Functionality test

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

Plant as Bioreactor

Production platform Cont

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.
Year 1989 Recombinant protein Mouse immunoglobulin Source Tobacco Maize Maize Importance 1st report on the production of antibody in plants. 1st commercialized plant-derived protein. 1st marketed plant-derived protein. 1st plant-based vaccine (for poultry) approved by the USDA 1st commercialized plant-derived antibody (marketed in Cuba) Reference Hiatt et al., 1989 Hood et al., 1997 Woodard et al., 2003 Dow AgroSciences, 2006

1997 Chicken avidin 2003 Bovine Trypsin HN proteins of

2006 Newcastle disease

virus 2007 Antibody against Hepatitis B

Rice

Tobacco

Pujol et al., 2007

Plant as Bioreactor

Production platforms (Cont.)

Legumes:
Soybean and pea: Seeds have high protein content (2040%). 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)

Plant as Bioreactor

Where they are produced?

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.

Plant as Bioreactor

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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 bioreactors Source: www.plantbioreactor.co.in/images/00_112.jpg

Plant as Bioreactor

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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.

Plant as Bioreactor

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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

Plant as Bioreactor

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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

Plant as Bioreactor

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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:
-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).

Plant as Bioreactor

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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).

Plant as Bioreactor

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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)

Plant as Bioreactor

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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.
Plant as Bioreactor

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Conclusion (Cont.)

Table source: Yansong miao et al., 2008

Plant as Bioreactor

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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.
Plant as Bioreactor

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References
Boothe JG, Parmenter DL, Saponja JA. (1997) Molecular farming in plants: oilseeds as vehicles for the production of

pharmaceutical proteins. Drug Dev Res, 42:17281.

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, 26:13018.

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: 10927.
Goldstein DA, Thomas JA. (2004), Biopharmaceuticals derived from genetically modified plants. QJM, 97: 70516. Hiatt A, Cafferkey R, Bowdish K. (1989), Production of antibodies in transgenic plants. Nature, 342: 768. 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: 2235262.
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: 291306.

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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: 50410.
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: 20715.
Lee S, An G. (2009), Over-expression of OsIRT1 leads to increased iron and zinc accumulations in rice. Plant Cell

Environ, 32: 40816.

Ma JK, Drake PM, Christou P. (2003), the production of recombinant pharmaceutical proteins in plants. Nat Rev Genet,

4: 794805.
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: 77627.

Nunes AC, Kalkmann DC, Arago 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, Ramrez NI, Ayala M, Gavilondo JV, Valds R, Rodrguez M, (2005), An integral approach towards a practical

application for a plant-made monoclonal antibody in vaccine purification. Vaccine, 23: 18337.

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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: 354651.
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: 77582.

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. 8:25563.

Sharma AK, Jani D, Tyagi AK. (2004), Transgenic plants as bioreactors. Ind J Biotechnol. 3: 27490. Streatfield SJ, Howard JA. (2003), Plant-based vaccines. Int J Parasitol, 33: 47993. Tiwari S, Verma PC, Singh PK, Tuli R. (2009), Plants as bioreactors for the production of vaccine antigens. Biotech

adv, 44967.
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: 12330.
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: 183945.

Plant as Bioreactor